Orally administered epigallocatechin gallate attenuates light-induced photoreceptor damage

Brain Research Bulletin 76 (2008) 412–423

Research report

Orally administered epigallocatechin gallate attenuates light-induced photoreceptor damage Belmira Lara da Silveira Andrade da Costa a , Rebecca Fawcett b , Guang-Yu Li c , Rukhsana Safa b , Neville N. Osborne b,∗ a

Departamento de Fisiologia e Farmacologia, CCB, Centro de Ciˆencias Biol´ogicas, CCB-UFPE, Avenue Prof. Moraes Rego, s/n, Cidade Universit´aria, 50670901 Recife, PE, Brazil b Nuffield Laboratory of Ophthalmology, University of Oxford, Walton Street, Oxford OX2 6AW, UK c The Second Hospital of Jilin University, Changchun 130041, China Received 6 November 2007; received in revised form 23 January 2008; accepted 31 January 2008 Available online 21 February 2008

Abstract EGCG, a major component of green tea, has a number of properties which includes it being a powerful antioxidant. The purpose of this investigation was to deduce whether inclusion of EGCG in the drinking water of albino rats attenuates the effect of a light insult (2200 lx, for 24 h) to the retina. TUNEL-positive cells were detected in the outer nuclear layer of the retina, indicating the efficacy of the light insult in inducing photoreceptor degeneration. Moreover, Ret-P1 and the mRNA for rhodopsin located at photoreceptors were also significantly reduced as well as the amplitude of both the a- and b-waves of the electroretinogram was also reduced showing that photoreceptors in particular are affected by light. An increase in protein/mRNA of GFAP located primarily to M¨uller cells caused by light shows that other retinal components are also influenced by the light insult. However, antigens associated with bipolar (␣-PKC), ganglion (Thy-1) and amacrine (GABA) cells, in contrast, appeared unaffected. The light insult also caused a change in the content of various proteins (caspase-3, caspase-8, PARP, Bad, and Bcl-2) involved in apoptosis. A number of the changes to the retina caused by a light insult were significantly attenuated when EGCG was in the drinking water. The reduction of the a- and b-waves and photoreceptor specific mRNAs/protein caused by light were significantly less. In addition, EGCG attenuated the changes caused by light to certain apoptotic proteins (especially at after 2 days) but did not appear to significantly influence the light-induced up-regulation of GFAP protein/mRNA. It is concluded that orally administered EGCG blunts the detrimental effect of light to the retina of albino rats where the photoreceptors are primarily affected. © 2008 Elsevier Inc. All rights reserved. Keywords: EGCG; Photoreceptor degeneration; Bad; Bcl-2; Caspase-3; Neuroprotection; ERG

1. Introduction A number of studies have shown that excessive visible light can induce photoreceptor and retinal pigment epithelial cell apoptosis in experimental animals [69,71,78,82]. However, it remains unclear whether this is due solely to a direct interaction of light with photoreceptors/retinal pigment epithelium chromophores or whether impaired choroidal circulation [69] and autophagy [37] caused by light are of significance. Light-induced photoreceptor degeneration may vary according to the light intensity [48], duration of light insult [40],

∗

Corresponding author. Tel.: +44 1865 248 996; fax: +44 1865 794 508. E-mail address: [email protected] (N.N. Osborne).

0361-9230/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2008.01.022

wavelength of light treatment [83], how animals were maintained before light treatment [58] and the time of day of light treatment [49]. The influence of light may also be reduced by limiting the rate of rhodopsin regeneration [29,62]. It is also apparent that reactive oxygen species (ROS) are generated in light-induced photoreceptor death which ultimately results in lipid peroxidation [82]. Oxidative stress is also recognised as an important factor in the pathophysiology of age-related macular degeneration and retinitis pigmentosa [1,2,67]. Moreover, antioxidant genes are up-regulated following photic injury [46,73] and light-induced photoreceptor cell death can be reduced by exogenous antioxidants such as ascorbate [36,52] and phenyl-N-tertbutylnitrone (PBN) [76] and various other substances [69,71,73]. The combination of various antioxidants is also able to delay the mutation dependent photorecep-

B.L. da Silveira Andrade da Costa et al. / Brain Research Bulletin 76 (2008) 412–423

tor degeneration in an animal model of retinitis pigmentosa [61]. Several studies have shown that certain plants, fruit and vegetables contain a variety of polyphenols that are known to act as antioxidants [12,66]. Important polyphenol compounds are the flavonoids which occur in varying amounts in different human diets [10]. Flavonoids are known to inhibit lipid peroxidation [47,74] and act as free radical scavengers [16,19,27]. A number of in vivo and in vitro reports have shown that catechins, a class of flavonoid present in large amounts in green tea, can exert neuroprotective actions in several models of neurodegenerative disorders [38,39,60]. The major catechin component of green tea, (−)-epigallocatechin-3-gallate (EGCG), at low micromolar and submicromolar concentrations attenuates a variety of insults by what appear to be different mechanisms. For example, EGCG acts as an antioxidant [4,43,84], increases endogenous antioxidant defences [21,30], counteracts inflammation-mediated neuronal injury [35] and causes a down-regulation of pro-apoptotic genes [25,81]. In relation to the eye, the antioxidant property of EGCG has been reported to account for its protective action of the lens against photooxidative stress [75,79,89]. Moreover, our recent studies have shown that photoreceptor degeneration induced by an intraocular injection of sodium nitroprusside (nitric oxide generator) can be attenuated by co-injection with EGCG [86] and that a systemic administration of EGCG attenuates retinal damage caused by ischemia/reperfusion [87]. These studies provided clear evidence that EGCG is a powerful antioxidant and can act as a neuroprotectant making its potential use for the treatment of retinal degenerative diseases worthy of consideration. Analysis of the bioavailability of EGCG in rats [41,65] shows orally administered EGCG to reach various tissues including the central nervous system, within 6 h. Moreover a second administration after a 6-hour interval enhances tissue levels 4–6 times above that of a single administration [65]. In addition, all the evidence suggests that when a large amount of EGCG is orally administered it is well tolerated with no ophthalmic abnormalities detected [22,23]. It has also been estimated that the half-life of green tea cathechins after high oral dosing is between 451 and 479 min in albino rats which is approximately 1–10 times longer than when administrated by an intravenous route [88]. In humans it has been shown that the pharmacokinetic parameters for EGCG are similar when present as decaffeinated green tea or as a pure form [33]. Oral administration of EGCG at a high daily dose of 800 mg is known to be safe and without side effects in healthy individuals [9]. The aim of the present studies was therefore specifically to deduce whether inclusion of EGCG in the drinking water of albino rats would be sufficient to attenuate the detrimental influence of an insult of constant light to their photoreceptors. 2. Methods 2.1. Animal care All procedures were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Home Office in the United Kingdom. Wistar rats were born and raised

413

Fig. 1. Levels of rhodopsin, NF-L, GFAP and caspase-3 mRNAs in retinas in control groups of rats where EGCG present or absent in the drinking water for a period of 7 days and maintained in normal animal house conditions. It can be seen that no difference was detected between both control groups of animals. under dim cyclic light (12-h light:12-h dark cycle) with food and water ad libitum before the experiments. Twenty-four animals (8 weeks of age) were used in the present study. The animals were subdivided into three groups (n = 8 per group): a control group maintained under dim cyclic light at all times and two groups submitted to a light-insult where EGCG was absent (light) or present in their drinking water (light + EGCG). In preliminary studies a group of six animals were treated with EGCG in the drinking water in the absence of light insult. No differences regarding biochemical, immunohistochemical and PCR analysis of the outer and inner retinas were detected between this group and the group where EGCG was not added to drinking water. Fig. 1 shows the level of rhodopsin, neurofilament (NFL), GFAP and caspase 3 mRNA levels in control groups of rats where EGCG was absent or present in their drinking water and maintained in normal animal house conditions.

2.2. Drug administration and exposure to light EGCG was kindly provided by Dr. Dario Rusciano (Sifi, Italy) and dissolved 0.4% in tap water. Two rats were placed in each open transparent animal cage (45.5 cm × 23 cm × 19 cm). Some cages were exposed to 2200 lx light intensity for 24 h. Environment temperature was monitored and maintained at 22 ◦ C. Also, EGCG was present in the drinking water of some rats which began 2 days before exposure and continued for another 4 days thereafter. During this period, each animal ingested about 17–20 ml of water per day whether EGCG was present or not. Thus, rats ingested on the average between 70 and 80 mg of EGCG per day or approximately 400 mg/kg body weight/day. This dose was based on published studies that determined the safety profile of EGCG consumption in albino rats [22,23]. A control group of rats not exposed to light received the same amount of EGCG in their drinking water (see Fig. 1). Animals were returned after light exposure to a 12-h dark:12-h light (50 lx) cycle.

2.3. Electroretinography Flash electroretinograms (ERGs) were recorded from both eyes of anesthetised rats 3 days before and 4 days after light exposure as previously described [6]. A few drops of 1% cyclopentolate hydrochloride (Nephew Pharmaceutical Ltd., Romford, UK) and a drop of the 0.4% benoxinate hydrochloride were applied to both eyes of rats in dim light in order to dilate their pupils and act as a topical anesthetic, respectively. The responses to a 2500 cd/m2 white light flash (1–2 s, 0.1 Hz) from a photic simulator (model PS33-plus; Grass-Telefactor, Instrument Division, West Warwick, UK) were placed at a distance of 50 cm were amplified and averaged using a 1902 signal Conditioner/1401 Laboratory Interface (CED, Cambridge, UK). The amplitude of the a-wave was measured from the baseline to the maximum a-wave trough and the b-wave was measured from the maximum a-wave trough to the maximum b-wave peak. Data were

414

B.L. da Silveira Andrade da Costa et al. / Brain Research Bulletin 76 (2008) 412–423

Table 1 RT-PCR primer sequences for mRNAs amplified mRNA

Primer Sequences

Annealing Temp (◦ C)

Accession Number

GAPDH

5 -CATCAAGAAGGTGGTGAAGCAGG-3

56

AF106860

Rhodopsin

5 -CAGTGTTCATGTGGGATTGACT-3 5 -ATGATTGGGTTGTAGATGGAGG-3

52

Z46957

GFAP

5 -ATTCCGCGCCTCTCCCTGTCTC-3 5 -GCTTCATCCGCCTCCTGTCTGT-3

55

U03700

NF-L

5 -ATGCTCAGATCTCCGTGGAGATG-3 5 -GCTTCGCAGCTCATTCTCCAGTT -3

52

AF031880

Caspase-3

5 -TCCCTCACCATTTCCTCTGGGCTGC-3 5 -ACTGGCTGCCCTCAGTTCCTGTGC-3

52

BC081854

Caspase-8

5 -ACTGGCTGCCCTCAAGTTCCTGTGC-3 5 -CCCTCACCATTTCCTCTGGGCTGC-3

60

AF279308

5 -CCACCACCCTGTTGCTGTAGCCA-3

analysed by the Student’s unpaired t-test and a p value of 0.05 was considered significant.

2.4. Immunohistochemistry Five days after light exposure, freshly dissected retinas were fixed in 2% paraformaldehyde in phosphate buffer saline (PBS) for 30 min and then washed in PBS + 0.3% triton (PBST), and cryopreserved in 30% sucrose in PBS. Frozen vertical retinal sections (10 ␮m) were then produced by using a cryostate. Retinal sections were incubated overnight at 4 ◦ C with rabbit monoclonal anti-GABA (1:500, Sigma, Poole UK) mouse monoclonal PKC-␣ (clone 165, 1:200, Sigma, Poole, UK), mouse anti-choline Thy-1 monoclonal (1:50; gift from Dunn School of Pathology, Oxford University) and developed with rabbit or mouse IgG conjugated to fluorescein-isothiocyanate (FITC).

2.5. The TUNEL procedure The procedure of Gavrieli et al. [14] was used to detect DNA fragmentation (TUNEL method) as an indication of apoptosis. Thawed frozen retinal sections were incubated for 5 min at room temperature in 30 mM Tris/HCl (pH 7.8). Incubation was continued at 37 ◦ C for 10 min in the presence of pepsin (800 U/ml) to expose the free DNA ends. After the pepsin was washed away with Tris/HCl, sections were preincubated for 10 min with buffer A (30 mM Tris/HCl, pH 7.2), 140 nM sodium cacodylate, and 1 mM cobalt chloride) and then placed in a humid chamber for 60 min at 37 ◦ C with buffer A containing 0.2 U/␮L terminal-deoxynucleotidyl transferase (TdT; Roche Diagnostics, Livinsgston, Scotland, UK) and 15 ␮M biotin-16-dUTP (Roche Diagnostics). The reaction was stopped by a 15-min wash in sodium citrate buffer (300 nM NaCl, 30 mM sodium citrate pH 7.3) at room temperature. Sections were then rinsed for 10 min in Tris-buffered saline (TBS: 20 mM tris, 150 mM NaCl, pH 7.3), for a further 10 min in TBS solution containing 1% bovine serum albumin and finally for 10-20 min with Cy-3 conjugated streptavidin. A final washing step was in TBS buffer. Sections were then mounted in buffered glycerol containing phenylenediamine to reduce fluorescence fading.

of 25 ng total RNA, PCR buffer, MgCl2 (5 mM for NFL, caspase-3, caspase8; 4.5 mM for GAPDH and 4 mM for all other primers), dNTPS, the relevant sense and anti-sense primer pairs and DNA polymerase. Reactions were initiated by incubating at 94 ◦ C for 10 min and PCRs (94 ◦ C, 15 s; 52 ◦ C, 55 ◦ C or 56 ◦ C, 30 s; 72 ◦ C, 30 s) performed for a suitable number of cycles followed by a final extension at 72 ◦ C for 3 min. Inter-experimental variations were avoided by performing all amplifications in a single run. The oligonucleotides primer pairs were obtained from Gibco Life Technologies (Paiseley, Scotland, UK) and their sequences, size, annealing temperatures and accession number are shown in Table 1. Prior to semi-quantitative amplification of the experimental samples, the amount of cDNA in all of the samples was equalized. In addition, the optimal conditions (e.g. Mg2+ concentration, annealing temperature) for each set of primers were determined. Subsequently, cycle-dependent reactions were performed for each mRNA amplified and the data were averaged. For assessment of the levels of the various mRNAs in the retina, all values were normalised to that of the housekeeping gene GAPDH that was used as an internal standard to correct for any variations in RNA isolation and/or cDNA synthesis. PCR reaction products were separated on 1.2% agarose gels using ethidium bromide for visualisation. The relative abundance of each PCR product was determined by quantitative analysis of digital photographs of gels using Labworks software (UVP Products, CA). RT, water/template controls were regularly included however in this case cDNA was excluded and substituted with water. The RT control did not contain reverse transcriptase (M-MLV). Fig. 2 shows an example gel containing the water and RT control as compared to GAPDH. All results are expressed as mean ± standard error of the mean (S.E.M.) and differences between control and treated retinas were assessed using Student’s paired t-test, while differences between treatment groups were assessed using Student’s unpaired t-test and a p value of 0.05 was considered significant.

2.6. Determination of mRNA levels by reverse transcriptase-PCR The levels of GAPDH, rhodopsin, neurofilament light form (NF-L; 70–80 kDa), glial fibrillary acidic protein (GFAP), caspase-3, caspase-8 mRNAs present in the retinas 5 days after light exposure were determined using a semi-quantitative reverse transcriptase-polymerase chain reaction technique (RT-PCR) as described previously [8,44]. Briefly, retinas were initially sonicated in Tri-reagent (Sigma, Poole, UK), the total RNA was isolated and first strand cDNA synthesis performed on 2 ␮g DNase-treated RNA. The individual cDNA species were amplified in a reaction containing a cDNA equivalent

Fig. 2. Representative gel containing water and RT control as compared to GAPDH. The water/template control was carried out according to the normal protocol; however the cDNA was substituted with water. The RT control was carried out as normal however the mix added to the normal mRNA did not contain reverse transcriptase (M-MLV). All three samples were amplified for 21 cycles.

B.L. da Silveira Andrade da Costa et al. / Brain Research Bulletin 76 (2008) 412–423

2.7. Protein extraction, electrophoresis and Western blotting In the retinal samples from eyes obtained 5 days after light exposure, proteins were isolated simultaneously with RNA. The proteins were then prepared with an initial wash with 100% ethanol, followed by three times washes with guanidine in 95% ethanol and left to dissolve in 20 mM Tris/HCl buffer (pH 7.4) containing 2 mM EDTA, 0.5 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride. Once fully dissolved equal volumes of sample buffer (62.5 mM Tris/HCl, pH 7.4, containing 4% SDS, 10% glycerol, 10% ␤-mercaptoethanol, and 0.002% bromophenol blue) was added, and samples were boiled for approximately 3 min and subjected to protein analysis by Western blotting. Retinal samples obtained 2 days after light exposure were treated only for Western blot analysis. In these cases, retinas were homogenized in freshly prepared 20 mM Tris/HCl buffer (pH 7.4) containing 2 mM EDTA, 0.5 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride. An equal volume of sample buffer (62.5 mM tris/HCl, pH 7.4, containing 4% SDS, 10% glycerol, 10% ␤-mercaptoethanol, and 0.002% bromophenol blue) was added, and samples were boiled for approximately 3 min. An aliquot was taken for determination of protein content. Fractioning of protein samples was achieved using 10% polyacrylamide gels containing 0.1% SDS. Samples were transferred onto nitrocellulose, as previously described [77]. The nitrocellulose blots were incubated with mouse anti-caspase-3 monoclonal (clone 46; 1:100; Becton-Dickenson, Cowley, Oxford), mouse anti-PARP (poly ADPribose polymerase; recognising the uncleaved and cleaved forms; 1:100; BD Pharmigen), mouse anti-Bcl-2 monoclonal (1:500; Santa Cruz Biotechnology, USA), rabbit anti-Bad polyclonal (1:1000, Cell Signaling Technology, USA) rabbit anti-actin (1:2000; Chemicon, Chandler’s Ford, UK) rabbit anti-rhodopsin kinase (1:1000; Affinity Bioreagents, Cambridge, UK), Ret-P1 (1:500, kind gift from Dr. C. J. Barnstable), anti-NF-L (1:1000; Chemicon) and then incubated for 3 h at room temperature. They were subsequently exposed to appropriate secondary antibodies conjugated to horseradish peroxidase and developed with a 0.016% solution of 3-amino-9-ethylcarbazole in 50 nM sodium acetate (pH 5) containing 0.05% Tween-20 and 0.03% H2 O2 . Digital images of the blots were obtained and the integrated optical density was estimated by using Labworks software (UVP Products, CA). The levels of the various proteins in the retinas were normalised to that of the actin protein that was used as an internal standard. The statistical analysis was done as described for mRNA levels by RT-PCR.

415

icantly attenuated by light treatment. Moreover, it is clear that the changes caused by light are less in rats where EGCG was present in their drinking water. Fig. 3B shows quantitative analysis of the a- and b-wave amplitudes from a number of animals. Light reduces the a- and b-wave amplitudes by approximately 70 and 65%, respectively. However, when EGCG is present in drinking water the reduction of both amplitudes is significantly less. 3.2. Histochemistry Fig. 4 shows a representative retinal section of the rat retina stained for the breakdown of DNA by the TUNEL procedure. In the control retina (Fig. 4A) TUNEL positive cells were completely absent. However sections of the retina from animals exposed to light 2 days earlier exhibited varying amounts of TUNEL-positive cells in different eccentricities (Fig. 4B and C). In contrast, very few TUNEL positive cells were generally present in all eccentricities of the retina from rats exposed to light 5 days earlier (Fig. 4D). No attempt was made to quantify the numbers of TUNEL-positive cells because of their variation in numbers and location. Fig. 5 shows retinal sections of approximately the same eccentricities from control rats and rats exposed to light 5 days earlier and stained for the localisation of ␣-PKC (Fig. 5A and D), GABA (Fig. 5B and E) or Thy-1 (Fig. 5C and F) immunoreactivities. Light had no obvious effect on the localisation and intensity of these antigens. On the other hand, GFAP immunoreactivity in M¨uller cells was increased by light such was unaffected when EGCG was present in the drinking water (Fig. 5G–I). 3.3. Western-blot data

3. Results 3.1. Electroretinography Fig. 3A shows that the normal a- and b-wave amplitudes of the scotopic ERG of the retina (baseline recording) are signif-

Fig. 6 shows that the protein level of Ret-P1 and rhodopsin kinase, relative to actin, are drastically reduced in retinas from animals exposed to light 2 or 5 days earlier. The reduction of Ret-P1 caused by light was significantly blunted in rats given EGCG in their drinking water (Fig. 6A and C). However, in

Fig. 3. (A) Representative ERG recordings of retinas from control rats (baseline) or rats given a light-insult where EGCG was absent (light) or present in their drinking water (EGCG + light). Light caused a reduction of the normal (baseline) a- and b-wave amplitudes which was significantly attenuated by EGCG. (B) Average values for a- and b-wave amplitudes from right and left retinas obtained for 8 animals per group. EGCG significantly attenuates the deleterious effect of light increasing the amplitude of both waves. Error bars represent S.E.M., where n = 16. ** p < 0.01, *** p < 0.001.

416

B.L. da Silveira Andrade da Costa et al. / Brain Research Bulletin 76 (2008) 412–423

and caspase-3 mRNAs (Fig. 9B and C) were increased by 200, 54 and 23%, respectively. However, the presence of EGCG in drinking water had no effect on the increases in mRNAs of these substances. In contrast, rhodopsin mRNA was significantly (about 30%) reduced by light (Fig. 9A) and EGCG attenuated this reduction by a small (about 10%) but significant amount (p = 0.032). 4. Discussion

Fig. 4. Representative retinal sections stained for TUNEL in control condition (A), 2 days after a light insult (B–C), or 5 days after a light insult (D). It can be seen that the insult has specifically caused TUNEL positive cells (arrows in B and C) to appear in the outer nuclear layer (ONL). Scale bars = 50 ␮m.

the case of rhodopsin kinase the presence of EGCG in drinking water was without effect (Fig. 6B and D). Light caused an up-regulation of caspase-3 (Fig. 7A), PARP (Fig. 7B) and Bad (Fig. 7C) but a down-regulation of Bcl-2 (Fig. 7D) in retinas analysed 2 days after light exposure. Importantly, EGCG significantly reduced the light-induced increase in caspase-3, Bad and PARP levels but did not modify the level of Bcl-2. Fig. 8A shows the protein levels for NF-L and GFAP in retinas 5 days after light exposure. Only GFAP is significantly increased by light but is unaffected when EGCG was present in the drinking water. 3.4. RT-PCR data NF-L mRNA (Fig. 8B) level was unaffected by light in retinas 5 days after a light insult while GFAP (Fig. 8B), caspase-8

The reported studies show that light-induced oxidative stress to the retina can be attenuated by EGCG, as occurs for other forms of oxidative stress to the retina, e.g. sodium nitroprusside [86] or ischemia/reperfusion [87]. Moreover, the finding that EGCG is effective when adminstered via the drinking water is important as it shows that the substance is able to reach the retina in sufficient amounts to have a positive physiological influence. This might be of clinical relevance for the treatment of ophthalmic disorders. It is known that EGCG can be consumed and tolerated at high doses [22,23]. In the present experiments we chose a defined insult based on the amount of light (24 h, 2200 lx) and the analysis time thereafter, to give a minimum measurable affect. Our unpublished information showed that when the insult is too pronounced, e.g. clear histological loss of photoreceptors, then reversing such an insult by pharmacological treatment is almost impossible. We are of the opinion, therefore, that if a substance has appropriate neuroprotective action then it should be effective in a model system where measured retinal damage is minimal. As a consequence we provide biochemical rather than histochemical evidence for EGCG attenuating the effect of a light insult to the retina. Our biochemical data show that some of the mRNAs and proteins that are affected by a light insult are blunted by EGCG but others are not. For example, rhodopsin and rhodopsin kinase mRNAs are significantly reduced by light but only rhodopsin mRNA is significantly less affected by EGCG treatment. We cannot, however, exclude the possibility that had analysis of mRNAs been undertaken at 2 or 8 days rather than 5 days after a light insult rhodopsin kinase mRNA may have been significantly affected by EGCG. No clear evidence was found to suggest that neurons other than photoreceptors are affected by the light insult used in this study. NF-L and Thy-1 antigens are primarily located to ganglion cells and NF-L protein/mRNA and retinal Thy-1 immunoreactivity are unaffected by light. Moreover, PKC-␣ and GABA immunoreactivities, located to rod bipolar cells and amacrine cells, respectively, were unaffected by the light insult. We therefore conclude that there is no evidence to suggest that the light insult used in this study affects any neuronal cell-type significantly other than photoreceptors. It is well known that an insult to the retina, including by light, causes gliosis and an elevation of GFAP expression in Muller cells [5,6,55]. It is also known that when microglia and/or M¨uller cells are activated they release various substances, some having neuroprotective functions [20,64] and others being detrimental [42]. For example, an ischemic preconditioning stimulus to the retina causes an increase in GFAP and bFGF, but it is thought

B.L. da Silveira Andrade da Costa et al. / Brain Research Bulletin 76 (2008) 412–423

417

Fig. 5. Immunohistochemical localisation of ␣-PKC (A and D), GABA (B and E) and Thy-1 (C and F) in representative retinas from control animals (A–C) or rats exposed to light (D–F). Representative GFAP immunoreactivity in control rats (G), rats exposed to light (H) and rats that ingested EGCG in the drinking water and were exposed to light (I). It can be seen that the immunoreactivity pattern for each antigen in the inner retina was not modified by light when compared with the control condition. Arrows in (A) and (D) indicate the presence of ␣-PKC immunoreactivity in on-bipolar cells, some amacrine cells and a more intense labelling of cell processes in the inner region of the inner plexiform layer (IPL). GABA-immunoreactivity is present in some cell bodies in the inner nuclear (INL) and ganglion cell layer (GCL) and in neuronal processes in the IPL. Thy-1 is located to ganglion bodies and their dendrites throughout the IPL and GCL with the ganglion cell axons in the nerve fibre layer being particularly strongly labelled. Light-induced expressive GFAP immunoreactivity in Muller cells (H) but this increase was not significantly reduced by EGCG. OPL = outer plexiform layer; ONL = outer nuclear layer. Scale bars = 50 ␮m.

that bFGF is the agent responsible for the attenuation of photoreceptor degeneration when the retina is given a subsequent insult of light [6]. The light-induced GFAP-immunoreactivity in Muller cells and the increase of GFAP protein/mRNA levels found in the present study therefore reflects retinal glial cell activation. Our studies also show that oral administration of EGCG did not counteract glial cell activation caused by light. Inclusion of EGCG in the drinking water of rats partially, but significantly, blunts the light-induced reduction of the aand b-wave amplitudes of the flash ERG. The a-wave reflects the functional integrity of the photoreceptors. The b-wave gives principally a measure of photoreceptor, ON-bipolar cells and M¨uller cell functions [56]. There is good reason to conclude that light directly affects photoreceptors. Changes in the flash ERG, following a light insult, is likely to arise from any alteration in the transduction process. Therefore photoreceptors are primarily affected either in terms of dysfunction or death (apoptosis or necrosis). It is clear from our biochemical data and TUNEL staining that only the photoreceptors are clearly dam-

aged by a light insult, suggesting this to be the major cause for the reduction in the a- and b-wave amplitudes. However, we cannot exclude the possibility that the change in the b-wave is partially due to M¨uller cells dysfunction which is suggested by an increase in GFAP activity. The current biochemical/histochemical studies clearly show, however, that light causes photoreceptor dysfunction/death. Apoptotic photoreceptors are clearly demonstrated by the TUNEL procedure and photoreceptor-specific rhodopsin mRNA and Ret-P1 protein are significantly reduced by light. When EGCG is present in the rats’ drinking water, the reduction of rhodopsin mRNA and Ret-P1 proteins caused by light is significantly less. Rhodopsin kinase, which is also photoreceptor specific, is also significantly affected but to a similar extent whether EGCG was present or absent from the drinking water. An explanation for this might be due to the fact that phosphorylation and consequent deactivation of rhodopsin by rhodopsin kinase is necessary to restore the dark state of the cell and to adjust the light sensitivity of the photoreceptor cell to different intensities of ambient illumination [28], and as a consequence any positive effect of EGCG is masked.

418

B.L. da Silveira Andrade da Costa et al. / Brain Research Bulletin 76 (2008) 412–423

Fig. 6. Protein levels of 39 kDa Ret-P1 (A and C), 60 kDa rhodopsin kinase (B and D) in retinal homogenates from eyes of controls or eyes analysed 2 days (A and B) or 5 days (C and D) after light exposure where EGCG was present or absent in the drinking water. EGCG was able to significantly blunt the reduction of Ret-P1 both at 2 and 5 days after light insult. However, this was not the case for rhodopsin kinase. Protein levels were normalised to the structural protein actin (42 kDa). Results are means ± S.E.M. where n = 12 for Ret-P1 and n = 16 for rhodopsin kinase. * p < 0.05, ** p < 0.01.

Accumulating evidence supports the hypothesis that exposure of the retina to intense light causes lipid peroxidation of retinal tissue. Lipid peroxidation can be propagated by free radicals and induces plasma membrane injury [50,68–70]. Although a complex defensive system composed of endogenous antioxidant enzymes can be activated after the light insult [46,85], if illumination persists, the excessive production of ROS overcomes this defensive mechanism causing an oxidative stress that can damage the photoreceptors. While the protective action of EGCG in the present studies cannot be attributed primarily to the antioxidant properties of the substance, logic suggests that this might be the case. EGCG has been shown to be 10 times more potent than vitamin E (trolox) at reducing lipid peroxidation of brain membranes [86] and there is impressive evidence to show that oxidative stress is involved in light-induced photoreceptor degeneration [35,69,73,76]. However, in vitro studies have also demonstrated that green tea polyphenols and flavonoids may induce activation of endogenous defence mechanisms that include specific transcription factors [21,30]. It is worth noting

here that intraperitoneal or oral administration of sulforaphane, an isothiocyanate found in some vegetables, has been shown to reduce the light-induced damage to the retina by increasing the expression of thioredoxin [72]. Other mechanisms for the potential action of EGCG include the modulation of signal transduction pathways, cell survival/death genes and enhanced mitochondrial function [38]. Mitochondria have been implied in the regulation of apoptosis in several types of pathologies [54,57]. Bad and Bax proteins opposite in their action to Bcl-2 can trigger the opening of the mitochondrial permeability transition pore (mPTP), or a specific channel in the outer mitochondrial membrane potential which can be involved in the release of pro-apoptotic factors from the mitochondria [3]. In the present study EGCG was able to attenuate the light-induced up-regulation of Bad but did not change the expression of Bcl-2. These results are in agreement with studies that show acute bright light exposure to cause a change in the mitochondrial membrane potential, which might be associated with the induction of photoreceptor apoptosis [11] and

B.L. da Silveira Andrade da Costa et al. / Brain Research Bulletin 76 (2008) 412–423

419

Fig. 7. Levels of 17 kDa caspase-3 (A), 89 kDa cleaved-PARP (B), 23 kDa Bad (C) and 17–20 kDa Bcl-2 (D) proteins in retinas from controls and 2 days after light where EGCG was present or absent in the drinking water. Light caused an up-regulation of caspase-3, Bad and PARP and a down-regulation of Bcl-2. EGCG significantly attenuated the effects of light on caspase-3, Bad and PARP proteins but did not modify the level of Bcl-2 protein. Protein levels were normalised to the structural protein actin (42 kDa). Results are means ± S.E.M. where n = 6. * p < 0.05, ** p < 0.01.

that the ablation of some pro-apoptotic Bcl-2 family members protected the retina against light damage [18]. Moreover, the present findings substantiate the view that EGCG has an action on pro-apoptotic gene cascades, as has been shown to occur in studies on a human neuroblastoma cell line [25,34,81]. In another in vivo study oral consumption of EGCG alone was shown to reduce Bax protein expression in dopaminergic neurons of the substantia nigra pars compacta and counteracted the increase of this protein induced by chemical insult in the same area [39]. Although in the present study EGCG was not able to attenuate the light-induced down-regulation in the levels of Bcl-2 protein, the decline in Bad expression may favour the

increase in the ratio of Bcl-2/Bad proteins, thereby contributing to mitochondrial stability and regulation of mPTP. In albino rats the light-induced oxidative process results in alkali-sensitive DNA damage as well as retinal DNA strand breaks. It was suggested that visible-light-induced damage occurs by a biphasic mechanism [63] and DNA repair occurs around 24 h after insult [15]. Antioxidant treatment indicated an early phase of ROS-mediated DNA damage, leading to a later stage of non-random enzymatic cleavage [32,45,50,52,63,71]. Several reports have indicated that visual-cell DNA losses were greatest during the first 3 days after a 24 h light exposure [40,51,63] and that the activation of caspase-3 may be one of the

420

B.L. da Silveira Andrade da Costa et al. / Brain Research Bulletin 76 (2008) 412–423

Fig. 8. Protein and mRNA levels in control retina and 5 days after a light insult. It can be seen that light has no influence on the NFL protein (A) or mRNA (B) in animals treated or not treated with EGCG. However, in the case of GFAP a significant increase in protein (A) and mRNA (B) was induced by light, but this was not affected in animals treated with EGCG. Protein and mRNA were normalised to the structural protein actin (42 kDa) and to GAPDH, respectively. Results are means ± S.E.M. where n = 16. * p < 0.05, ** p < 0.01.

Fig. 9. Levels of rhodopsin (A), caspase-8 (B) and caspase-3 (C) mRNAs in retinas 5 days after light exposure compared with controls. It can be seen that EGCG significantly attenuated the light-induced decrease of rhodopsin mRNA, but did not affect the up-regulation of caspase-8 or caspase-3. In each case the level of mRNA was related to the mRNA of GAPDH. Results are means ± S.E.M. where n = 16. * p < 0.05, ** p < 0.01.

B.L. da Silveira Andrade da Costa et al. / Brain Research Bulletin 76 (2008) 412–423

final executioners of photoreceptor cell death following photic injury in vivo [59,82]. Caspase-3 mediates apoptosis by cleaving several targets, including the nuclear enzyme PARP [31] that plays a pivotal role in apoptosis in several experimental models of photoreceptor degeneration [7,13]. The ability of EGCG to significantly reduce the expression of cleaved caspase-3 and PARP, 2 days after light exposure, indicates its neuroprotective action in light-induced apoptotic cell death in the early stages after insult. Nevertheless, cell death of the photoreceptor population following photic injury lasts several days and a progressive reduction in the ONL thickness was reported even 3 months after insult [17,71]. Evidence of antioxidant protection in the later stages of photoreceptor damage was obtained by using ascorbate [50,52,53], TEMPOL [71,80], DMTU [32,48], PBN [76] among other potent antioxidants [26,69,73]. In the present study, 5 days after light insult, only a few TUNEL positive cells were detected in the outer nuclear layer, and a significant increase in the protein levels of Ret-P1 and mRNA levels of rhodopsin was induced by EGCG. However, at this time point, the lightinduced up-regulation of caspase-8 and caspase-3 mRNAs was not significantly reduced by EGCG. It is possible that in addition to photoreceptors, the light insult was able to induce activation of caspase-3 in other retinal cell types. Future studies are required to address this issue and would be important to analyse the effects of increasing concentrations of EGCG in the water or the maintenance of the oral treatment over a longer time period. In conclusion, the results obtained in the present study show that oral intake of EGCG can reach the retina and attenuate light-induced photoreceptor degeneration in rats. These studies support the notion that daily oral intake of EGCG might benefit patients suffering from retinal diseases such as glaucoma [24] and age-related macular degeneration [67] where oxidative stress is implicated to occur. Acknowledgments We are grateful to Dr. Dario Rusciano (Sifi, Italy) who provided a sample of EGCG and to Nigel Swietalski for excellent technical assistance. Guang-Yu Li was sponsored by China Scholarship Council and Jilin University, China, and Belmira Lara da S. A. da Costa received a Scholarship from Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq, Brazil). References [1] J. Ambati, B.K. Ambati, S.H. Yoo, S. Ianchulev, A.P. Adamis, Age-related macular degeneration: etiology, pathogenesis, and therapeutic strategies, Surv. Ophthalmol. 48 (2003) 257–293. [2] S. Beatty, H. Koh, M. Phil, D. Henson, M. Boulton, The role of oxidative stress in the pathogenesis of age-related macular degeneration, Surv. Ophthalmol. 45 (2000) 115–134. [3] P. Bernardi, V. Petronilli, F. Di Lisa, M. Forte, A mitochondrial perspective on cell death, Trends Biochem. Sci. 26 (2001) 112–117. [4] R. Buttemeyer, A.W. Philipp, L. Schlenzka, J.W. Mall, M. Beissenhirtz, F. Lisdat, Epigallocatechin gallate can significantly decrease free oxygen radicals in the reperfusion injury in vivo, Transplant. Proc. 35 (2003) 3116–3120.

421

[5] W. Cao, F. Li, R.H. Steinberg, M.M. Lavail, Development of normal and injury-induced gene expression of aFGF, bFGF, CNTF, BDNF GFAP and IGF-I in the rat retina, Exp. Eye Res. 72 (2001) 591–604. [6] R.J. Casson, J.P. Wood, J. Melena, G. Chidlow, N.N. Osborne, The effect of ischemic preconditioning on light-induced photoreceptor injury, Invest. Ophthalmol. Vis. Sci. 44 (2003) 1348–1354. [7] C.J. Chang, C.H. Cherng, W.S. Liou, C.L. Liao, Minocycline partially inhibits caspase-3 activation and photoreceptor degeneration after photic injury, Ophthalmic. Res. 37 (2005) 202–213. [8] G. Chidlow, R. Casson, P. Sobrado-Calvo, M. Vidal-Sanz, N.N. Osborne, Measurement of retinal injury in the rat after optic nerve transection: an RT-PCR study, Mol. Vis. 11 (2005) 387–396. [9] S.H.-H. Chow, Y. Cai, I.A. Hakim, J.A. Crowell, F. Shahi, C.A. Brooks, R.T. Dorr, Y. Hara, D.S. Alberts, Pharmacokinetics and safety of green tea polyphenols after multiple-dose administration of epigallocatechin gallate and polyphenon E in healthy individuals, Clin. Cancer Res. 9 (2003) 3312–3319. [10] F. Dajas, F. Rivera-Megret, F. Blasina, F. Arredondo, J.A. Abin-Carriquiry, G. Costa, C. Echeverry, L. Lafon, H. Heizen, M. Ferreira, A. Morquio, Neuroprotection by flavonoids, Braz. J. Med. Biol. Res. 36 (2003) 1613– 1620. [11] M. Donovan, R.J. Carmody, T.G. Cotter, Light-induced photoreceptor apoptosis in vivo requires neuronal nitric-oxide synthase and guanylate cyclase activity and is caspase-3-independent, J. Biol. Chem. 276 (2001) 23000–23008. [12] E. Esposito, D. Rotilio, V. Di Matteo, C. Di Giulio, M. Cacchio, S. Algeri, A review of specific dietary antioxidants and the effects on biochemical mechanisms related to neurodegenerative processes, Neurobiol. Aging 23 (2002) 719–735. [13] R.J. Fawcett, N.N. Osborne, Flupirtine attenuates sodium nitroprussideinduced damage to retinal photoreceptors, in situ, Brain Res. Bull. 73 (2007) 278–288. [14] Y. Gavrieli, Y. Sherman, S.A. Ben-Sasson, Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation, J. Cell Biol. 119 (1992) 493–501. [15] W.C. Gordon, D.M. Casey, W.J. Lukiw, N.G. Bazan, DNA damage and repair in light-induced photoreceptor degeneration, Invest. Ophthalmol. Vis. Sci. 43 (2002) 3511–3521. [16] Q. Guo, B. Zhao, M. Li, S. Shen, W. Xin, Studies on protective mechanisms of four components of green tea polyphenols against lipid peroxidation in synaptosomes, Biochim. Biophys. Acta 1304 (1996) 210–222. [17] F. Hafezi, A. Marti, K. Munz, C.E. Reme, Light-induced apoptosis: differential timing in the retina and pigment epithelium, Exp. Eye Res. 64 (1997) 963–970. [18] P. Hahn, T. Lindsten, A. Lyubarsky, G.S. Ying, E.N. Pugh Jr., C.B. Thompson, J.L. Dunaief, Deficiency of Bax and Bak protects photoreceptors from light damage in vivo, Cell Death Differ. 11 (2004) 1192–1197. [19] Y. Hanasaki, S. Ogawa, S. Fukui, The correlation between active oxygens scavenging and antioxidative effects of flavonoids, Free Radic. Biol. Med. 16 (1994) 845–850. [20] T. Harada, C. Harada, N. Nakayama, S. Okuyama, K. Yoshida, S. Kohsaka, H. Matsuda, K. Wada, Modification of glial-neuronal cell interactions prevents photoreceptor apoptosis during light-induced retinal degeneration, Neuron 26 (2000) 533–541. [21] J.V. Higdon, B. Frei, Tea catechins and polyphenols: health effects, metabolism, and antioxidant functions, Crit. Rev. Food Sci. Nutr. 43 (2003) 89–143. [22] R.A. Isbrucker, J. Bausch, J.A. Edwards, E. Wolz, Safety studies on epigallocatechin gallate (EGCG) preparations. Part 1: genotoxicity, Food Chem. Toxicol. 44 (2006) 626–635. [23] R.A. Isbrucker, J.A. Edwards, E. Wolz, A. Davidovich, J. Bausch, Safety studies on epigallocatechin gallate (EGCG) preparations. Part 2: dermal, acute and short-term toxicity studies, Food Chem. Toxicol. 44 (2006) 636–650. [24] A. Izzotti, A. Bagnis, S.C. Sacca, The role of oxidative stress in glaucoma, Mutat. Res. 612 (2006) 105–114. [25] L. Kalfon, M.B. Youdim, S.A. Mandel, Green tea polyphenol (−) epigallocatechin-3-gallate promotes the rapid protein kinase C- and

422

[26]

[27] [28] [29]

[30]

[31] [32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

B.L. da Silveira Andrade da Costa et al. / Brain Research Bulletin 76 (2008) 412–423 proteasome-mediated degradation of Bad: implications for neuroprotection, J. Neurochem. 100 (2007) 992–1002. I. Kaldi, M. Dittmar, P. Pierce, R.E. Anderson, l-NAME protects against acute light damage in albino rats, but not against retinal degeneration in P23H and S334ter transgenic rats, Exp. Eye Res. 76 (2003) 453–461. M. Kashima, Effects of catechins on superoxide and hydroxyl radical, Chem. Pharm. Bull. (Tokyo) 47 (1999) 279–283. U.B. Kaupp, R. Seifert, Cyclic nucleotide-gated ion channels, Physiol. Rev. 82 (2002) 769–824. C. Keller, C. Grimm, A. Wenzel, F. Hafezi, C. Reme, Protective effect of halothane anesthesia on retinal light damage: inhibition of metabolic rhodopsin regeneration, Invest. Ophthalmol. Vis. Sci. 42 (2001) 476–480. M.R. Kelly, C.M. Geigerman, G. Loo, Epigallocatechin gallate protects U937 cells against nitric oxide-induced cell cycle arrest and apoptosis, J. Cell Biochem. 81 (2001) 647–658. D.W. Koh, T.M. Dawson, V.L. Dawson, Mediation of cell death by poly (ADP-ribose) polymerase-1, Pharmacol. Res. 52 (2005) 5–14. S. Lam, M.O. Tso, D.H. Gurne, Amelioration of retinal photic injury in albino rats by dimethylthiourea, Arch. Ophthalmol. 108 (1990) 1751– 1757. M.J. Lee, P. Maliakal, L. Chen, X. Meng, F.Y. Bondoc, S. Prabhu, G. Lambert, S. Mohr, C.S. Yang, Pharmacokinetics of tea catechins after ingestion of green tea and (−)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability, Cancer Epidemiol. Biomarkers Prev. 11 (2002) 1025–1032. Y. Levites, T. Amit, M.B. Youdim, S. Mandel, Involvement of protein kinase C activation and cell survival/cell cycle genes in green tea polyphenol (−)-epigallocatechin 3-gallate neuroprotective action, J. Biol. Chem. 277 (2002) 30574–30580. R. Li, Y.G. Huang, D. Fang, W.D. Le, (−)-Epigallocatechin gallate inhibits lipopolysaccharide-induced microglial activation and protects against inflammation-mediated dopaminergic neuronal injury, J. Neurosci. Res. 78 (2004) 723–731. Z.Y. Li, M.O. Tso, H.M. Wang, D.T. Organisciak, Amelioration of photic injury in rat retina by ascorbic acid: a histopathologic study, Invest. Ophthalmol. Vis. Sci. 26 (1985) 1589–1598. H.R. Lohr, K. Kuntchithapautham, A.K. Sharma, B. Rohrer, Multiple, parallel cellular suicide mechanisms participate in photoreceptor cell death, Exp. Eye Res. 83 (2006) 380–389. S.A. Mandel, Y. Avramovich-Tirosh, L. Reznichenko, H. Zheng, O. Weinreb, T. Amit, M.B. Youdim, Multifunctional activities of green tea catechins in neuroprotection. Modulation of cell survival genes, iron-dependent oxidative stress and PKC signaling pathway, Neurosignals 14 (2005) 46–60. S. Mandel, O. Weinreb, T. Amit, M.B. Youdim, Cell signaling pathways in the neuroprotective actions of the green tea polyphenol (−)-epigallocatechin-3-gallate: implications for neurodegenerative diseases, J. Neurochem. 88 (2004) 1555–1569. M. Moriya, B.N. Baker, T.P. Williams, Progression and reversibility of early light-induced alterations in rat retinal rods, Cell Tissue Res. 246 (1986) 607–621. K. Nakagawa, T. Miyazawa, Absorption and distribution of tea catechin, (−)-epigallocatechin-3-gallate, in the rat, J. Nutr. Sci. Vitaminol. (Tokyo) 43 (1997) 679–684. T. Nakazawa, M. Takeda, G.P. Lewis, K.S. Cho, J. Jiao, U. Wilhelmsson, S.K. Fisher, M. Pekny, D.F. Chen, J.W. Miller, Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin, Invest. Ophthalmol. Vis. Sci. 48 (2007) 2760–2768. F. Nanjo, K. Goto, R. Seto, M. Suzuki, M. Sakai, Y. Hara, Scavenging effects of tea catechins and their derivatives on 1,1-diphenyl-2picrylhydrazyl radical, Free Radic. Biol. Med. 21 (1996) 895–902. M.S. Nash, N.N. Osborne, Assessment of Thy-1 mRNA levels as an index of retinal ganglion cell damage, Invest. Ophthalmol. Vis. Sci. 40 (1999) 1293–1298. W.K. Noell, D.T. Organisciak, H. Ando, M.A. Braniecki, C. Durlin, Ascorbate and dietary protective mechanisms in retinal light damage of rats: electrophysiological, histological and DNA measurements, Prog. Clin. Biol. Res. 247 (1987) 469–483.

[46] A. Ohira, M. Tanito, S. Kaidzu, T. Kondo, Glutathione peroxidase induced in rat retinas to counteract photic injury, Invest. Ophthalmol. Vis. Sci. 44 (2003) 1230–1236. [47] J. Okuda, I. Miwa, K. Inagaki, T. Horie, M. Nakayama, Inhibition of aldose reductases from rat and bovine lenses by flavonoids, Biochem. Pharmacol. 31 (1982) 3807–3822. [48] D.T. Organisciak, R.M. Darrow, L. Barsalou, R.A. Darrow, R.K. Kutty, G. Kutty, B. Wiggert, Light history and age-related changes in retinal light damage, Invest. Ophthalmol. Vis. Sci. 39 (1998) 1107–1116. [49] D.T. Organisciak, R.M. Darrow, L. Barsalou, R.K. Kutty, B. Wiggert, Circadian-dependent retinal light damage in rats, Invest. Ophthalmol. Vis. Sci. 41 (2000) 3694–3701. [50] D.T. Organisciak, R.M. Darrow, Y.I. Jiang, G.E. Marak, J.C. Blanks, Protection by dimethylthiourea against retinal light damage in rats, Invest. Ophthalmol. Vis. Sci. 33 (1992) 1599–1609. [51] D.T. Organisciak, Y.L. Jiang, H.M. Wang, M. Pickford, J.C. Blanks, Retinal light damage in rats exposed to intermittent light: comparison with continuous light exposure, Invest. Ophthalmol. Vis. Sci. 30 (1989) 795–805. [52] D.T. Organisciak, H.M. Wang, Z.Y. Li, M.O. Tso, The protective effect of ascorbate in retinal light damage of rats, Invest. Ophthalmol. Vis. Sci. 26 (1985) 1580–1588. [53] D.T. Organisciak, A. Xie, H.M. Wang, Y.L. Jiang, R.M. Darrow, L.A. Donoso, Adaptive changes in visual cell transduction protein levels: effect of light, Exp. Eye Res. 53 (1991) 773–779. [54] S. Orrenius, Mitochondrial regulation of apoptotic cell death, Toxicol. Lett. 149 (2004) 19–23. [55] N.N. Osborne, F. Block, K.H. Sontag, Reduction of ocular blood flow results in glial fibrillary acidic protein (GFAP) expression in rat retinal M¨uller cells, Vis. Neurosci. 7 (1991) 637–639. [56] N.N. Osborne, R.J. Casson, J.P. Wood, G. Chidlow, M. Graham, J. Melena, Retinal ischemia: mechanisms of damage and potential therapeutic strategies, Prog. Retin. Eye Res. 23 (2004) 91–147. [57] N.N. Osborne, G. Lascaratos, A.J. Bron, G. Chidlow, J.P. Wood, A hypothesis to suggest that light is a risk factor in glaucoma and the mitochondrial optic neuropathies, Br. J. Ophthalmol. 90 (2006) 237–241. [58] J.S. Penn, M.I. Naash, R.E. Anderson, Effect of light history on retinal antioxidants and light damage susceptibility in the rat, Exp. Eye Res. 44 (1987) 779–788. [59] O. Perche, M. Doly, I. Ranchon-Cole, Caspase-dependent apoptosis in light-induced retinal degeneration, Invest. Ophthalmol. Vis. Sci. 48 (2007) 2753–2759. [60] L. Reznichenko, T. Amit, M.B. Youdim, S. Mandel, Green tea polyphenol (−)-epigallocatechin-3-gallate induces neurorescue of long-term serumdeprived PC12 cells and promotes neurite outgrowth, J. Neurochem. 93 (2005) 1157–1167. [61] M.M. Sanz, L.E. Johnson, S. Ahuja, P.A. Ekstrom, J. Romero, T. van Veen, Significant photoreceptor rescue by treatment with a combination of antioxidants in an animal model for retinal degeneration, Neuroscience 145 (2007) 1120–1129. [62] P.A. Sieving, P. Chaudhry, M. Kondo, M. Provenzano, D. Wu, T.J. Carlson, R.A. Bush, D.A. Thompson, Inhibition of the visual cycle in vivo by 13-cis retinoic acid protects from light damage and provides a mechanism for night blindness in isotretinoin therapy, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 1835–1840. [63] S. Specht, M. Leffak, R.M. Darrow, D.T. Organisciak, Damage to rat retinal DNA induced in vivo by visible light, Photochem. Photobiol. 69 (1999) 91–98. [64] J. Stone, J. Maslim, K. Valter-Kocsi, K. Mervin, F. Bowers, Y. Chu, N. Barnett, J. Provis, G. Lewis, S.K. Fisher, S. Bisti, C. Gargini, L. Cervetto, S. Merin, J. Peer, Mechanisms of photoreceptor death and survival in mammalian retina, Prog. Retin. Eye Res. 18 (1999) 689–735. [65] M. Suganuma, S. Okabe, M. Oniyama, Y. Tada, H. Ito, H. Fujiki, Wide distribution of [3H](−)-epigallocatechin gallate, a cancer preventive tea polyphenol, in mouse tissue, Carcinogenesis 19 (1998) 1771–1776. [66] H. Sung, J. Nah, S. Chun, H. Park, S.E. Yang, W.K. Min, In vivo antioxidant effect of green tea, Eur. J. Clin. Nutr. 54 (2000) 527–529.

B.L. da Silveira Andrade da Costa et al. / Brain Research Bulletin 76 (2008) 412–423 [67] M. Suzuki, M. Kamei, H. Itabe, K. Yoneda, H. Bando, N. Kume, Y. Tano, Oxidized phospholipids in the macula increase with age and in eyes with age-related macular degeneration, Mol. Vis. 13 (2007) 772–778. [68] M. Tanito, H. Haniu, M.H. Elliott, A.K. Singh, H. Matsumoto, R.E. Anderson, Identification of 4-hydroxynonenal-modified retinal proteins induced by photooxidative stress prior to retinal degeneration, Free Radic. Biol. Med. 41 (2006) 1847–1859. [69] M. Tanito, S. Kaidzu, R.E. Anderson, Delayed loss of cone and remaining rod photoreceptor cells due to impairment of choroidal circulation after acute light exposure in rats, Invest. Ophthalmol. Vis. Sci. 48 (2007) 1864–1872. [70] M. Tanito, Y.W. Kwon, N. Kondo, J. Bai, H. Masutani, H. Nakamura, J. Fujii, A. Ohira, J. Yodoi, Cytoprotective effects of geranylgeranylacetone against retinal photooxidative damage, J. Neurosci. 25 (2005) 2396–2404. [71] M. Tanito, F. Li, M.H. Elliott, M. Dittmar, R.E. Anderson, Protective effect of TEMPOL derivatives against light-induced retinal damage in rats, Invest. Ophthalmol. Vis. Sci. 48 (2007) 1900–1905. [72] M. Tanito, H. Masutani, Y.C. Kim, M. Nishikawa, A. Ohira, J. Yodoi, Sulforaphane induces thioredoxin through the antioxidant-responsive element and attenuates retinal light damage in mice, Invest. Ophthalmol. Vis. Sci. 46 (2005) 979–987. [73] M. Tanito, H. Masutani, H. Nakamura, S. Oka, A. Ohira, J. Yodoi, Attenuation of retinal photooxidative damage in thioredoxin transgenic mice, Neurosci. Lett. 326 (2002) 142–146. [74] J. Terao, M. Piskula, Q. Yao, Protective effect of epicatechin, epicatechin gallate, and quercetin on lipid peroxidation in phospholipid bilayers, Arch. Biochem. Biophys. 308 (1994) 278–284. [75] G. Thiagarajan, S. Chandani, C.S. Sundari, S.H. Rao, A.V. Kulkarni, D. Balasubramanian, Antioxidant properties of green and black tea, and their potential ability to retard the progression of eye lens cataract, Exp. Eye Res. 73 (2001) 393–401. [76] H. Tomita, Y. Kotake, R.E. Anderson, Mechanism of protection from lightinduced retinal degeneration by the synthetic antioxidant phenyl-N-tertbutylnitrone, Invest. Ophthalmol. Vis. Sci. 46 (2005) 427–434.

423

[77] H. Towbin, T. Staehelin, J. Gordon, Electrophortic transfer of proteins from polyacrilamide gels to nitrocellulose sheets: procedure and some applications, Prog. Natl. Acad. Sci. U.S.A. 76 (1979) 4350–4354. [78] M. Tytell, M.F. Barbe, D.J. Gower, Photoreceptor protection from light damage by hyperthermia, Prog. Clin. Biol. Res. 314 (1989) 523–538. [79] J.A. Vinson, J. Zhang, Black and green teas equally inhibit diabetic cataracts in a streptozotocin-induced rat model of diabetes, J. Agric. Food Chem. 53 (2005) 3710–3713. [80] M. Wang, T.T. Lam, J. Fu, M.O. Tso, TEMPOL, a superoxide dismutase mimic, ameliorates light-induced retinal degeneration, Res. Commun. Mol. Pathol. Pharmacol. 89 (1995) 291–305. [81] O. Weinreb, S. Mandel, M.B. Youdim, Gene and protein expression profiles of anti- and pro-apoptotic actions of dopamine, R-apomorphine, green tea polyphenol (−)-epigallocatechine-3-gallate, and melatonin, Ann. N. Y. Acad. Sci. 993 (2003) 351–361. [82] A. Wenzel, C. Grimm, M. Samardzija, C.E. Reme, Molecular mechanisms of light-induced photoreceptor apoptosis and neuroprotection for retinal degeneration, Prog. Retin. Eye Res. 24 (2005) 275–306. [83] T.P. Williams, W.L. Howell, Action spectrum of retinal light-damage in albino rats, Invest. Ophthalmol. Vis. Sci. 24 (1983) 285–287. [84] D. Xie, G. Liu, G. Zhu, W. Wu, S. Ge, (−)-Epigallocatechin-3-gallate protects cultured spiral ganglion cells from H2O2-induced oxidizing damage, Acta Otolaryngol. 24 (2004) 464–470. [85] M. Yamamoto, K. Lidia, H. Goug, S. Onitsuka, T. Kotani, A. Ohira, Changes in manganese superoxide dismutase expression after exposure of the retina to intense light, Histochem. J. 31 (1999) 81–87. [86] B. Zhang, N.N. Osborne, Oxidative-induced retinal degeneration is attenuated by epigallocatechin gallate, Brain Res. 1124 (2006) 176–187. [87] B. Zhang, R. Safa, D. Rusciano, N.N. Osborne, Epigallocatechin gallate, an active ingredient from green tea, attenuates damaging influences to the retina caused by ischemia/reperfusion, Brain Res. 1159 (2007) 40– 53. [88] M. Zhu, Y. Chen, R.C. Li, Oral absorption and bioavailability of tea catechins, Planta Med. 66 (2000) 444–447. [89] S. Zigman, N.S. Rafferty, K.A. Rafferty, N. Lewis, Effects of green tea polyphenols on lens photooxidative stress, Biol. Bull. 197 (1999) 285–286.