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Neuroprotective effects of Epigallocatechin-3-gallate (EGCG) in optic nerve crush model in rats
Neuroscience Letters 479 (2010) 26–30
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Neuroprotective effects of Epigallocatechin-3-gallate (EGCG) in optic nerve crush model in rats Jun Xie a,1 , Libin Jiang a,∗,1 , Ting Zhang a , Yulan Jin b , Dongmei Yang b , Fei Chen a a Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Lab, 1# Dong Jiao Min Xiang, Beijing 100730, PR China b Pathology Department, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, PR China
a r t i c l e
i n f o
Article history: Received 18 April 2010 Accepted 7 May 2010 Keywords: Epigallocatechin-3-gallate (EGCG) Neuroprotection Retinal ganglion cells (RGCs) Optic nerve crush (ONC)
a b s t r a c t Epigallocatechin-3-gallate (EGCG), the major catechin found in green tea, is a powerful antioxidant and has anti-inflammatory with neuroprotective potential. This study aims to investigate the neuroprotective effects of EGCG in an optic nerve crush (ONC) model in rats. Seventy-two Wistar rats were randomly divided into four groups: normal control (group A), sham operation + EGCG (group B), ONC + vehicle (group C), and ONC + EGCG (group D). The rats were treated intraperitoneally and orally with either vehicle or EGCG (25 mg/kg, injected daily for 5 days and 2 mg/kg orally daily afterwards). Two days after the first injection, an ONC injury was performed by using a micro optic nerve clipper with 40 g power at approximately 2 mm from the optic nerve head for 60 s. Fluorogold was injected into the bilateral superior colliculi 5 days before sacrifice and fluorescent gold-labelled retinal ganglion cells (RGCs) were counted under fluorescence microscopy on days 7, 14 and 28 after ONC. The expression of Neurofilament triplet L (NF-L) was measured via immunohistochemical and Western blotting analysis. In group C, a progressive loss of RGCs was observed after ONC. In contrast, the density of RGCs was significantly higher in group D (p = 0.009, independent samples t-test) on day 7 after ONC, and statistical differences were obtained on days 14 and 28 (p = 0.026 and p = 0.019, respectively, independent samples t-test). The results of immunohistochemical and Western blotting analysis showed significantly higher NF-L protein expression in group D in comparison with group C on days 7, 14 and 28 after ONC. These findings suggest that there are protective effects of EGCG on RGCs after ONC, indicating EGCG might be a potential therapeutic agent for optic nerve diseases. © 2010 Elsevier Ireland Ltd. All rights reserved.
Optic nerve damage, caused by glaucoma and other optic nerve diseases, is a major cause of severe visual loss and blindness worldwide. Unfortunately, treatment to protect retinal ganglion cells (RGCs) and their axons in optic nerve diseases has not yet been established. Recent research has shown that epigallocatechin-3gallate (EGCG), a major polyphenol extracted from green tea, has a potential neuroprotective effect [12]. For example, in vivo studies have shown that EGCG prevents apoptosis in RGC-5 cells caused by H2 O2 and light [14,15]. Moreover, studies have demonstrated that sufficient amount of EGCG when delivered by intraperitoneally or orally can reach the retina and protect RGCs on day 7 after ischemia/reperfusion [9,14,15].
∗ Corresponding author at: Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, PR China. Tel.: +86 10 5826 9920; fax: +86 10 5826 9920. E-mail address: [email protected] (L. Jiang). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.05.020
It has been suggested that some loss of RGCs in glaucoma and other optic neuropathies is also due to indirect or secondary degeneration [2,4,7,10,13]. Given EGCG’s proven effects on neuroprotection, we explored whether EGCG is capable of preventing secondary degeneration of RGCs. This study aims to evaluate the neuroprotective effects of EGCG on RGCs in optic nerve crush (ONC) model, a well-known model for secondary degeneration of optic nerves [4,13]. Experiments were conducted on 72 female Wistar rats (7 weeks old, weighing 170–200 g). The animal protocol was approved by the University Institutional Animal Care and Use Committee of Capital Medical University and is consistent with the NIH Guide for the Care and Use of Laboratory Animals (National Institute of Health Publications, No. 80–23, revised 1996). All procedures were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The animals were randomly divided (randomized littermates) into four groups (n = 18 for each group): normal control (group A), sham operation + EGCG (group B), ONC + vehicle (group C), ONC + EGCG (group D). Within each group, rats were separately observed for 7, 14 and 28 days
J. Xie et al. / Neuroscience Letters 479 (2010) 26–30
after the ONC injury (n = 6 for each observation period). Group A, a normal control group, was used to evaluate the normal RGC density in the retina. In groups B and D, EGCG (from Sigma Chemical Co., St. Louis, MO) was injected intraperitoneally (25 mg/kg, dissolved in saline, once per day) for 5 days from 2 days before ONC to 2 days after ONC, and subsequently administered orally (2 mg/kg/day) until the end of the observation. These procedures were performed with slight modifications as described in previous research [11,15]. In group C, the animals received the vehicle instead (saline injection and oral feeding with drinking water). All manipulations were performed with the animals under general anaesthesia, achieved by intraperitoneal injection of 7% chloral hydrate solution (400 mg/kg), and using topical 0.5% Alcaine eye drops (Alcon, Puurs, Belgium). In groups C and D, ONC was performed 2 days after the first injection. Under an operating microscope, the lateral canthus was incised and the retractor bulbi muscle was separated, followed by blunt exposure of the optic nerve. Care was taken to avoid damaging the small vessels around the optic nerve. The optic nerve was then crushed using a micro optic nerve clipper (AS-1, 40 g force, width of the clipper head 1.5 mm, Japan) at approximately 2 mm from the optic nerve head for 60 s to ensure a reproducible injury on each animal. In the sham operation group, the same procedure was performed except ONC. After removing the clipper, examination of the fundus with a direct ophthalmoscope was performed to confirm the presence of blood perfusion and animals with interrupted blood supply were excluded from the study. The wound was then sutured and 0.3% ofloxacin ophthalmic ointment was instilled (Tarivid; Santen, Osaka, Japan). Five days prior to the scheduled sacrifice, each rat was anaesthetized and placed in a stereotactic apparatus. The skull was exposed and kept dry and clean. The bregma was identified and marked. The following four designated coordinates (corresponding to the bilateral superior colliculi) were marked on the skull: 5.9 mm and 6.4 mm behind the bregma, on the anteroposterior axis, 1.4 ± 0.2 mm lateral to the midline on both hemispheres. Two small windows (approximately 2 mm × 3 mm) were drilled in the skull on both hemispheres to allow injection of 1.5 L of 3% Fluoro-Gold (FG, Biotium Corporation, USA) in 0.9% saline at the coordinates described previously, using a Hamilton syringe (Hamilton, Reno, NV, USA). The tip of the syringe was advanced perpendicular to the skull by approximately 4.0 mm into the cortex during each injection, and after each injection, the syringe was kept unmoved for 5 min. The wound was then sutured, and antibiotic ointment was applied. Five days after FG labelling, the rats were transcardially perfused under deep anaesthesia with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2), at a flow rate of 20 ml/min. The eyes were enucleated and the 12 o’clock position was marked on the limbus. The globes were bisected at the equator with the lenses removed and the posterior segments were post-fixed for another 30 min with 4% paraformaldehyde. To prepare the flatmounts, the retina of an enucleated eye was dissociated from the underlying structures (sclera/choroid). Four radial cuts (superonasal, inferonasal, inferotemporal, and superotemporal) were made on the peripheral retina, and the retina was spread on a gelatin-coated glass slide. FG-labelled RGCs were examined under a fluorescence microscope (LEICA DM 4000B, Germany) with a UV filter (blue-violet: 395–440 nm). Fluorescence micrographs (200× magnification) were taken from each quadrant of the retina at a distance of 2 mm from the optic disc centre. Cell counting (cells/mm2 ) was performed by two investigators in a masked fashion and the results were averaged. To determine whether there was significant interobserver variation, 20 fields were counted independently by two masked observers. The Pearson correlation coefficient between the two observers was 0.96.
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Fixed optic nerves were embedded in paraffin and cut in longitudinal sections at 3 m. Retinal sections were first dewaxed, then subjected to retrieval for 2.5 min in 10 mmol/L of citrate buffer at pH 6.0 in an autoclave sterilizer, and incubated with 3% hydrogen peroxide for 10 min at room temperature to inhibit endogenous peroxidase activity. The slides were then incubated with a primary antibody of rabbit anti-neurofilament L (1:1000, Chemicon, USA) at 37 ◦ C for 2 h. This was followed by incubation with the corresponding secondary antibody. Haematoxylin-DAB was used for staining, resulting in a brown or dark brown signal. All of the sections were evaluated by two independent histopathology experts in a masked fashion. Western blot analysis was performed with modifications as described [15]. Protein samples containing 30 mg of protein were separated on 10% polyacrylamide gels containing 0.1% SDS and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated in TBST buffer (0.02 M Tris-base, pH 7.6, 0.8% NaCl, 0.1% Tween 20) supplemented with 5% dry skimmed milk for 60 min to block non-specific binding. The primary antibody of rabbit anti-neurofilament L (1:1000, Chemicon, USA), or actin (1:1000, Santa Cruz, USA) was added and the preparations were incubated at 4 ◦ C overnight and the appropriate secondary antibodies (goat anti-rabbit IgG conjugated to horseradish peroxidase) were subsequently used at room temperature for 1 h. Finally, immunoblotting signals were visualized by using the ECL-plus Kit. Quantification was performed on computer (Image J software). The RGC count was expressed as the mean ± standard deviation (95% confidence interval, CI) (Table 1). All measurements in this study were performed in a masked fashion. The data were analyzed for significance using the independent samples t-test, and RGC survival rates (compared with the fellow eye) in different groups were analyzed with a Chi-square test, using the SPSS 15.0 statistical package. A two-tailed p value of less than 0.05 was considered statistically significant. In group A (normal control), the mean RGC densities were 2465 ± 162 (95% CI: 2295–2634) cells/mm2 , 2486 ± 97 (95% CI: 2383–2588) cells/mm2 , and 2595 ± 160 (95% CI: 2427–2763) cells/mm2 , respectively at different time points. The mean RGC densities in group B (sham operation + EGCG) were 2535 ± 163 (95% CI: 2364–2706) cells/mm2 , 2507 ± 83 (95% CI: 2419–2594) cells/mm2 , and 2572 ± 150 (95% CI: 2415–2729) cells/mm2 , respectively at days 7 (n = 6), 14 (n = 6) and 28 (n = 6) after the procedure. Independent samples t-test showed that the mean RGC densities were not significantly different between group A and group B (p > 0.05). Furthermore, no time-dependent differences were found in the mean RGC densities. The results indicated that the operation and the drug used did not significantly impact the RGCs. The mean RGC density in group C (ONC + vehicle) on day 7 was 944 ± 85 (95% CI: 854–1033) cells/mm2 , and it decreased to 813 ± 172 (95% CI: 632–993) cells/mm2 on day 14, and to 767 ± 171 (95% CI: 587–947) cells/mm2 on day 28 after the crush procedure, showing a progressive loss of RGCs. This indicated that in the vehicle-treated rats, the mid-peripheral RGC density showed a survival rate of 38.3%, 32,7% and 29.6% at day 7, 14 and 28 after ONC, respectively. There was also progressive reduction in the density of RGCs with time (Table 1; Figs. 1 and 2). In group D (ONC + EGCG), the mean RGC densities were 1134 ± 118 (CI: 1011–1258) cells/mm2 , 1022 ± 94 (CI: 923–1120) cells/mm2 , and 1010 ± 126 (CI: 877–1142) cells/mm2 on days 7 (n = 6), 14 (n = 6) and 28 (n = 6), respectively. Compared with group C (ONC + vehicle), the severity of RGC loss in group D (ONC + EGCG) was significantly lower (p = 0.009, independent samples t-test) on day 7, and results on day 14 (p = 0.026, independent samples ttest) and day 28 (p = 0.019, independent samples t-test). The results demonstrated that the RGC survival rate increased to 44.75% on day 7, 40.76% on day 14 and 39.25% on day 28 in the EGCG-treated
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J. Xie et al. / Neuroscience Letters 479 (2010) 26–30
Table 1 FG-labelled RGCs densities on days 7, 14 and 28 after ONC in various groups. Observation period
Group A (N)
Group B (sham + EGCG)
Group C (ONC + vehicle)
Group D (ONC + EGCG)
6 944 ± 85 854–1033 38.28
6 1134 ± 118** 1011–1258 44.75***
Day 7 n Mean ± SD CI % survival
6 2465 ± 162 2295–2634
6 2535 ± 163 2364–2706
Day 14 n Mean ± SD CI % survival
6 2486 ± 97 2383–2588
6 2507 ± 83 2419–2594
6 813 ± 172 632–993 32.70
6 1022 ± 94* 923–1120 40.76***
Day 28 n Mean ± SD CI % survival
6 2595 ± 160 2427–2763
6 2572 ± 150 2415–2729
6 767 ± 171 587–947 29.55
6 1010 ± 126* 877–1142 39.25***
Note: RGCs densities are expressed in cells/mm2 , data are given as Mean ± SD. CI, confidence interval at 95% level. N, normal control; sham, sham operation; ONC, optic nerve crush. * p<0.05 (independent samples t-test), versus group C. ** p<0.01 (independent samples t-test), versus group C. *** p<0.001 (Chi-square test), versus group C.
Fig. 1. Fluorescence micrographs from representative regions of the rat retina on days 7, 14 and 28 after ONC, taken approximately 2 mm from the optic nerve head. (A) Group A (normal control); (B) group B (sham operation + EGCG); (C) group C (ONC + vehicle); (D) group D (ONC + EGCG). Scale bars indicate 20 m. (1) day 7; (2) day 14; (3) day 28.
J. Xie et al. / Neuroscience Letters 479 (2010) 26–30
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Fig. 2. RGC count in various groups on days 7, 14 and 28 after ONC. (A) Mean RGC count (cells/mm2 ) of various groups on days 7, 14 and 28. (B) RGC survival rates in groups C and D. Group A (normal control); group B (sham operation + EGCG); group C (ONC + vehicle); group D (ONC + EGCG).
group as compared to the vehicle-treated group (p < 0.001 for all three time points, i.e., days 7, 14 and 28, Chi-square test). These suggested the potential protective role of EGCG on RGCs (Table 1; Figs. 1 and 2). Normal NF-L staining in the optic nerve of rats is evenly distributed in the whole longitudinal section of the optic nerve. Faint staining for NF-L could be observed at 7, 14 and 28 days after ONC. In contrast, in the EGCG-treated optic nerves, the reduction in NF-L immunoreactivity was less pronounced (Fig. 3). The levels of NF-L protein in the optic nerve relative to actin are shown in Fig. 4. The results showed that the ONC significantly decreased the level of NF-L. In contrast, the effects of the crush were attenuated by EGCG treatment. Compared with the vehicle-treated group, a significant increase of the NF-L protein level was observed on days 7 (p < 0.01, independent samples t-test; Fig. 4), 14 (p < 0.01, independent samples t-test; Fig. 4) and 28 (p < 0.05, independent samples t-test; Fig. 4) after ONC. It is widely accepted that neuronal loss following nerve injury is greater than what might be expected from the severity of the injury and this is attributable to a variety of processes leading collectively to secondary degeneration [2,4,7,10,13]. Therefore, the term
neuroprotection now commonly refers to the protection of neurons which, following acute insult, do not sustain direct injury, but are adjacent to or surrounded by a damaged milieu and will consequently undergo “secondary degeneration” unless adequately treated [2,4,7,10,13]. In the rat ONC model, there is both direct and indirect damage of the neurones. While direct injury was considered to be the cause of massive cell loss soon after acute injury, indirect toxicity was believed to be responsible for sequential cell death [4,8,13] The loss of RGCs in the first week after the optic nerve injury is mainly due to primary (or direct) damage, and that in week 2 through week 4 after the injury is mainly caused by secondary (or indirect) degeneration [8,13]. In our study, we performed retrograde labelling of RGCs to quantify the number of RGCs at different times following ONC (on days 7, 14 and 28) and conducted immunohistochemistry and Western blot analyses to measure the expression of NF-L proteins in the axons of RGCs. While retrograde labelling of RGCs offers an objective measurement of RGCs survival, the expression of NF-L protein, one subunit of neurofilaments (NFs), is a viable measure of RGC viability [5]. The findings of our study demonstrate that EGCG could increase both the population of survived RGCs and the
Fig. 3. Immunohistochemistry of NF-L in the optic nerve. The optic nerve slices (3 m) were cut in longitudinal sections, then immunostained with rabbit anti-NF-L and restained with haematoxylin to show the distribution of NF-L (dark yellow) and the cell nucleus (blue), respectively. (A) Normal control; (B) negative control; (C) day 7 after ONC with vehicle; (D) day 7 after ONC with EGCG; (E) day 14 after ONC with vehicle; (F) day 14 after ONC with EGCG; (G) day 28 after ONC with vehicle; (H) day 28 after ONC with EGCG. Scale bars indicate 50 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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J. Xie et al. / Neuroscience Letters 479 (2010) 26–30
EGCG prevented secondary degeneration of RGCs. Therefore, how the multi-functions of RGC protection by EGCG play out would be a worthy direction in further investigations. Future research should also look into the protective effects of EGCG beyond week 4 after ONC. Acknowledgments This work was supported by the National Natural Science Foundation of China (grant no. 30973268). The authors thank Professor Timothy Lai at The Chinese University of Hong Kong for advice and critical comments. References
Fig. 4. Western blot analysis of NF-L (A) protein levels relative to actin of the optic nerves in the non-crush group (normal control) and days 7, 14 and 28 after ONC with vehicle (vehicle) or EGCG (EGCG), respectively. Quantitative analysis indicates a significant increment in NF-L expression with EGCG treatment on days 7, 14 and 28 after ONC (B). n = 3 in each case, *p < 0.05, **p < 0.01.
expression of NF-L in optic nerve on days 7, 14 and 28 after ONC. These results suggest that EGCG is capable of reducing both primary and secondary degeneration of RGCs, indicating that EGCG might be a treatment option for acute or chronic optic nerve diseases. Our study also demonstrated that EGCG may protect RGCs even in a small dose. Previous in vivo studies suggested that EGCG at the dose level of 50 mg/kg/day injected intraperitoneally or daily oral administration of 200 ml 0.5% EGCG is capable of protecting RGCs and photoreceptor cells [1,14,15]. Building on recent research on EGCG’ impact on cerebral ischemic damage [6] and aging-associated oxidative damage in rat brain [11], our study confirmed that EGCG is effective of neuroprotection even in a dose as low as 25 mg/kg/day injected intraperitoneally and 2 mg/kg/day given orally. EGCG has been proved to have a wide variety of properties, including antioxidant and free radical scavenging, antiinflammatory, and attenuating glutamate-induced cytotoxicity [12]. Secondary degeneration of RGCs involves mechanisms such as glutamate-induced excitotoxicity leading to calcium overload and oxidative stress, free radical formation, mitochondrial dysfunction and energy failure, enzymatic degradation, membrane instability and resultant inflammation [3]. Our study is limited in the sense that it could not reveal the exact mechanisms in which
[1] B.L.d.S.A.d. Costa, R. Fawcett, G.-Y. Li, R. Safa, N.N. Osborne, Orally administered epigallocatechin gallate attenuates light-induced photoreceptor damage, Brain Res. Bull. 76 (2008) 412–423. [2] A. Faden, Pharmacological treatment of central nervous system trauma, Pharmacol. Toxicol. 18 (1996) 12–17. [3] M. Fitzgerald, C.A. Bartlett, L. Evill, J. Rodger, A.R. Harvey, S.A. Dunlop, Secondary degeneration of the optic nerve following partial transection: the benefits of lomerizine, Exp. Neurol. 216 (2009) 219–230. [4] T.V. Johnson, S.I. Tomarev, Rodent models of glaucoma, Brain Res. Bull. 81 (2010) 349–358. [5] M.K. Lee, D.W. Cleveland, Neurofilament function and dysfunction: involvement in axonal growth and neuronal disease, Curr. Opin. Cell Biol. 6 (1994) 34–40. [6] S. Lee, S. Suh, S. Kim, Protective effects of the green tea (−)-epigallocatechin gallate against hippocampal neuronal damage after transient global ischemia in gerbils, Neurosci. Lett. 28 (2000) 191–194. [7] H. Levkovitch-Verbin, R. Dardik, S. Vander, S. Melamed, Mechanism of retinal ganglion cells death in secondary degeneration of the optic nerve, Exp. Eye Res. (2009) (available online 29.11.09). [8] H. Levkovitch-Verbin, H.A. Quigley, K.R. Martin, D.J. Zack, M.E. Pease, D.F. Valenta, A model to study differences between primary and secondary degeneration of retinal ganglion cells in rats by partial optic nerve transaction, Invest. Ophthalmol. Vis. Sci. 44 (2003) 3388–3393. [9] P. Peng, M. Ko, C. Chen, Epigallocatechin-3-gallate reduces retinal ischemia/reperfusion injury by attenuating neuronal nitric oxide synthase expression and activity, Exp. Eye Res. 86 (2008) 637–646. [10] D.H. Smith, K. Casey, T.K. McIntosh, Pharmacologic therapy for traumatic brain injury: experimental approaches, New Horiz. 3 (1995) 562–572. [11] R. Srividhya, V. Jyothilakshmi, K. Arulmathi, V. Senthilkumaran, P. Kalaiselvi, Attenuation of senescence-induced oxidative exacerbations in aged rat brain by (−)-epigallocatechin-3-gallate, Int. J. Dev. Neurosci. 26 (2008) 217–223. [12] B.A. Sutherland, R.M.A. Rahman, I. Appleton, Mechanisms of action of green tea catechins, with a focus on ischemia-induced neurodegeneration, J. Nutr. Biochem. 17 (2006) 291–306. [13] E Yoles, M. Schwartz, Degeneration of spared axons following partial white matter lesion: implications for optic nerve neuropathies, Exp. Neurol. 153 (1998) 1–7. [14] B. Zhang, D. Rusciano, N.N. Osborne, Orally administered epigallocatechin gallate attenuates retina neuronal death in vivo and light-induced apoptosis in vitro, Brain Res. 1198 (2008) 141–152. [15] 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.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Neuroprotective effects of Epigallocatechin-3-gallate (EGCG) in optic nerve crush model in rats Jun Xie a,1 , Libin Jiang a,∗,1 , Ting Zhang a , Yulan Jin b , Dongmei Yang b , Fei Chen a a Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Lab, 1# Dong Jiao Min Xiang, Beijing 100730, PR China b Pathology Department, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, PR China
a r t i c l e
i n f o
Article history: Received 18 April 2010 Accepted 7 May 2010 Keywords: Epigallocatechin-3-gallate (EGCG) Neuroprotection Retinal ganglion cells (RGCs) Optic nerve crush (ONC)
a b s t r a c t Epigallocatechin-3-gallate (EGCG), the major catechin found in green tea, is a powerful antioxidant and has anti-inflammatory with neuroprotective potential. This study aims to investigate the neuroprotective effects of EGCG in an optic nerve crush (ONC) model in rats. Seventy-two Wistar rats were randomly divided into four groups: normal control (group A), sham operation + EGCG (group B), ONC + vehicle (group C), and ONC + EGCG (group D). The rats were treated intraperitoneally and orally with either vehicle or EGCG (25 mg/kg, injected daily for 5 days and 2 mg/kg orally daily afterwards). Two days after the first injection, an ONC injury was performed by using a micro optic nerve clipper with 40 g power at approximately 2 mm from the optic nerve head for 60 s. Fluorogold was injected into the bilateral superior colliculi 5 days before sacrifice and fluorescent gold-labelled retinal ganglion cells (RGCs) were counted under fluorescence microscopy on days 7, 14 and 28 after ONC. The expression of Neurofilament triplet L (NF-L) was measured via immunohistochemical and Western blotting analysis. In group C, a progressive loss of RGCs was observed after ONC. In contrast, the density of RGCs was significantly higher in group D (p = 0.009, independent samples t-test) on day 7 after ONC, and statistical differences were obtained on days 14 and 28 (p = 0.026 and p = 0.019, respectively, independent samples t-test). The results of immunohistochemical and Western blotting analysis showed significantly higher NF-L protein expression in group D in comparison with group C on days 7, 14 and 28 after ONC. These findings suggest that there are protective effects of EGCG on RGCs after ONC, indicating EGCG might be a potential therapeutic agent for optic nerve diseases. © 2010 Elsevier Ireland Ltd. All rights reserved.
Optic nerve damage, caused by glaucoma and other optic nerve diseases, is a major cause of severe visual loss and blindness worldwide. Unfortunately, treatment to protect retinal ganglion cells (RGCs) and their axons in optic nerve diseases has not yet been established. Recent research has shown that epigallocatechin-3gallate (EGCG), a major polyphenol extracted from green tea, has a potential neuroprotective effect [12]. For example, in vivo studies have shown that EGCG prevents apoptosis in RGC-5 cells caused by H2 O2 and light [14,15]. Moreover, studies have demonstrated that sufficient amount of EGCG when delivered by intraperitoneally or orally can reach the retina and protect RGCs on day 7 after ischemia/reperfusion [9,14,15].
∗ Corresponding author at: Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, PR China. Tel.: +86 10 5826 9920; fax: +86 10 5826 9920. E-mail address: [email protected] (L. Jiang). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.05.020
It has been suggested that some loss of RGCs in glaucoma and other optic neuropathies is also due to indirect or secondary degeneration [2,4,7,10,13]. Given EGCG’s proven effects on neuroprotection, we explored whether EGCG is capable of preventing secondary degeneration of RGCs. This study aims to evaluate the neuroprotective effects of EGCG on RGCs in optic nerve crush (ONC) model, a well-known model for secondary degeneration of optic nerves [4,13]. Experiments were conducted on 72 female Wistar rats (7 weeks old, weighing 170–200 g). The animal protocol was approved by the University Institutional Animal Care and Use Committee of Capital Medical University and is consistent with the NIH Guide for the Care and Use of Laboratory Animals (National Institute of Health Publications, No. 80–23, revised 1996). All procedures were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The animals were randomly divided (randomized littermates) into four groups (n = 18 for each group): normal control (group A), sham operation + EGCG (group B), ONC + vehicle (group C), ONC + EGCG (group D). Within each group, rats were separately observed for 7, 14 and 28 days
J. Xie et al. / Neuroscience Letters 479 (2010) 26–30
after the ONC injury (n = 6 for each observation period). Group A, a normal control group, was used to evaluate the normal RGC density in the retina. In groups B and D, EGCG (from Sigma Chemical Co., St. Louis, MO) was injected intraperitoneally (25 mg/kg, dissolved in saline, once per day) for 5 days from 2 days before ONC to 2 days after ONC, and subsequently administered orally (2 mg/kg/day) until the end of the observation. These procedures were performed with slight modifications as described in previous research [11,15]. In group C, the animals received the vehicle instead (saline injection and oral feeding with drinking water). All manipulations were performed with the animals under general anaesthesia, achieved by intraperitoneal injection of 7% chloral hydrate solution (400 mg/kg), and using topical 0.5% Alcaine eye drops (Alcon, Puurs, Belgium). In groups C and D, ONC was performed 2 days after the first injection. Under an operating microscope, the lateral canthus was incised and the retractor bulbi muscle was separated, followed by blunt exposure of the optic nerve. Care was taken to avoid damaging the small vessels around the optic nerve. The optic nerve was then crushed using a micro optic nerve clipper (AS-1, 40 g force, width of the clipper head 1.5 mm, Japan) at approximately 2 mm from the optic nerve head for 60 s to ensure a reproducible injury on each animal. In the sham operation group, the same procedure was performed except ONC. After removing the clipper, examination of the fundus with a direct ophthalmoscope was performed to confirm the presence of blood perfusion and animals with interrupted blood supply were excluded from the study. The wound was then sutured and 0.3% ofloxacin ophthalmic ointment was instilled (Tarivid; Santen, Osaka, Japan). Five days prior to the scheduled sacrifice, each rat was anaesthetized and placed in a stereotactic apparatus. The skull was exposed and kept dry and clean. The bregma was identified and marked. The following four designated coordinates (corresponding to the bilateral superior colliculi) were marked on the skull: 5.9 mm and 6.4 mm behind the bregma, on the anteroposterior axis, 1.4 ± 0.2 mm lateral to the midline on both hemispheres. Two small windows (approximately 2 mm × 3 mm) were drilled in the skull on both hemispheres to allow injection of 1.5 L of 3% Fluoro-Gold (FG, Biotium Corporation, USA) in 0.9% saline at the coordinates described previously, using a Hamilton syringe (Hamilton, Reno, NV, USA). The tip of the syringe was advanced perpendicular to the skull by approximately 4.0 mm into the cortex during each injection, and after each injection, the syringe was kept unmoved for 5 min. The wound was then sutured, and antibiotic ointment was applied. Five days after FG labelling, the rats were transcardially perfused under deep anaesthesia with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2), at a flow rate of 20 ml/min. The eyes were enucleated and the 12 o’clock position was marked on the limbus. The globes were bisected at the equator with the lenses removed and the posterior segments were post-fixed for another 30 min with 4% paraformaldehyde. To prepare the flatmounts, the retina of an enucleated eye was dissociated from the underlying structures (sclera/choroid). Four radial cuts (superonasal, inferonasal, inferotemporal, and superotemporal) were made on the peripheral retina, and the retina was spread on a gelatin-coated glass slide. FG-labelled RGCs were examined under a fluorescence microscope (LEICA DM 4000B, Germany) with a UV filter (blue-violet: 395–440 nm). Fluorescence micrographs (200× magnification) were taken from each quadrant of the retina at a distance of 2 mm from the optic disc centre. Cell counting (cells/mm2 ) was performed by two investigators in a masked fashion and the results were averaged. To determine whether there was significant interobserver variation, 20 fields were counted independently by two masked observers. The Pearson correlation coefficient between the two observers was 0.96.
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Fixed optic nerves were embedded in paraffin and cut in longitudinal sections at 3 m. Retinal sections were first dewaxed, then subjected to retrieval for 2.5 min in 10 mmol/L of citrate buffer at pH 6.0 in an autoclave sterilizer, and incubated with 3% hydrogen peroxide for 10 min at room temperature to inhibit endogenous peroxidase activity. The slides were then incubated with a primary antibody of rabbit anti-neurofilament L (1:1000, Chemicon, USA) at 37 ◦ C for 2 h. This was followed by incubation with the corresponding secondary antibody. Haematoxylin-DAB was used for staining, resulting in a brown or dark brown signal. All of the sections were evaluated by two independent histopathology experts in a masked fashion. Western blot analysis was performed with modifications as described [15]. Protein samples containing 30 mg of protein were separated on 10% polyacrylamide gels containing 0.1% SDS and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated in TBST buffer (0.02 M Tris-base, pH 7.6, 0.8% NaCl, 0.1% Tween 20) supplemented with 5% dry skimmed milk for 60 min to block non-specific binding. The primary antibody of rabbit anti-neurofilament L (1:1000, Chemicon, USA), or actin (1:1000, Santa Cruz, USA) was added and the preparations were incubated at 4 ◦ C overnight and the appropriate secondary antibodies (goat anti-rabbit IgG conjugated to horseradish peroxidase) were subsequently used at room temperature for 1 h. Finally, immunoblotting signals were visualized by using the ECL-plus Kit. Quantification was performed on computer (Image J software). The RGC count was expressed as the mean ± standard deviation (95% confidence interval, CI) (Table 1). All measurements in this study were performed in a masked fashion. The data were analyzed for significance using the independent samples t-test, and RGC survival rates (compared with the fellow eye) in different groups were analyzed with a Chi-square test, using the SPSS 15.0 statistical package. A two-tailed p value of less than 0.05 was considered statistically significant. In group A (normal control), the mean RGC densities were 2465 ± 162 (95% CI: 2295–2634) cells/mm2 , 2486 ± 97 (95% CI: 2383–2588) cells/mm2 , and 2595 ± 160 (95% CI: 2427–2763) cells/mm2 , respectively at different time points. The mean RGC densities in group B (sham operation + EGCG) were 2535 ± 163 (95% CI: 2364–2706) cells/mm2 , 2507 ± 83 (95% CI: 2419–2594) cells/mm2 , and 2572 ± 150 (95% CI: 2415–2729) cells/mm2 , respectively at days 7 (n = 6), 14 (n = 6) and 28 (n = 6) after the procedure. Independent samples t-test showed that the mean RGC densities were not significantly different between group A and group B (p > 0.05). Furthermore, no time-dependent differences were found in the mean RGC densities. The results indicated that the operation and the drug used did not significantly impact the RGCs. The mean RGC density in group C (ONC + vehicle) on day 7 was 944 ± 85 (95% CI: 854–1033) cells/mm2 , and it decreased to 813 ± 172 (95% CI: 632–993) cells/mm2 on day 14, and to 767 ± 171 (95% CI: 587–947) cells/mm2 on day 28 after the crush procedure, showing a progressive loss of RGCs. This indicated that in the vehicle-treated rats, the mid-peripheral RGC density showed a survival rate of 38.3%, 32,7% and 29.6% at day 7, 14 and 28 after ONC, respectively. There was also progressive reduction in the density of RGCs with time (Table 1; Figs. 1 and 2). In group D (ONC + EGCG), the mean RGC densities were 1134 ± 118 (CI: 1011–1258) cells/mm2 , 1022 ± 94 (CI: 923–1120) cells/mm2 , and 1010 ± 126 (CI: 877–1142) cells/mm2 on days 7 (n = 6), 14 (n = 6) and 28 (n = 6), respectively. Compared with group C (ONC + vehicle), the severity of RGC loss in group D (ONC + EGCG) was significantly lower (p = 0.009, independent samples t-test) on day 7, and results on day 14 (p = 0.026, independent samples ttest) and day 28 (p = 0.019, independent samples t-test). The results demonstrated that the RGC survival rate increased to 44.75% on day 7, 40.76% on day 14 and 39.25% on day 28 in the EGCG-treated
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Table 1 FG-labelled RGCs densities on days 7, 14 and 28 after ONC in various groups. Observation period
Group A (N)
Group B (sham + EGCG)
Group C (ONC + vehicle)
Group D (ONC + EGCG)
6 944 ± 85 854–1033 38.28
6 1134 ± 118** 1011–1258 44.75***
Day 7 n Mean ± SD CI % survival
6 2465 ± 162 2295–2634
6 2535 ± 163 2364–2706
Day 14 n Mean ± SD CI % survival
6 2486 ± 97 2383–2588
6 2507 ± 83 2419–2594
6 813 ± 172 632–993 32.70
6 1022 ± 94* 923–1120 40.76***
Day 28 n Mean ± SD CI % survival
6 2595 ± 160 2427–2763
6 2572 ± 150 2415–2729
6 767 ± 171 587–947 29.55
6 1010 ± 126* 877–1142 39.25***
Note: RGCs densities are expressed in cells/mm2 , data are given as Mean ± SD. CI, confidence interval at 95% level. N, normal control; sham, sham operation; ONC, optic nerve crush. * p<0.05 (independent samples t-test), versus group C. ** p<0.01 (independent samples t-test), versus group C. *** p<0.001 (Chi-square test), versus group C.
Fig. 1. Fluorescence micrographs from representative regions of the rat retina on days 7, 14 and 28 after ONC, taken approximately 2 mm from the optic nerve head. (A) Group A (normal control); (B) group B (sham operation + EGCG); (C) group C (ONC + vehicle); (D) group D (ONC + EGCG). Scale bars indicate 20 m. (1) day 7; (2) day 14; (3) day 28.
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Fig. 2. RGC count in various groups on days 7, 14 and 28 after ONC. (A) Mean RGC count (cells/mm2 ) of various groups on days 7, 14 and 28. (B) RGC survival rates in groups C and D. Group A (normal control); group B (sham operation + EGCG); group C (ONC + vehicle); group D (ONC + EGCG).
group as compared to the vehicle-treated group (p < 0.001 for all three time points, i.e., days 7, 14 and 28, Chi-square test). These suggested the potential protective role of EGCG on RGCs (Table 1; Figs. 1 and 2). Normal NF-L staining in the optic nerve of rats is evenly distributed in the whole longitudinal section of the optic nerve. Faint staining for NF-L could be observed at 7, 14 and 28 days after ONC. In contrast, in the EGCG-treated optic nerves, the reduction in NF-L immunoreactivity was less pronounced (Fig. 3). The levels of NF-L protein in the optic nerve relative to actin are shown in Fig. 4. The results showed that the ONC significantly decreased the level of NF-L. In contrast, the effects of the crush were attenuated by EGCG treatment. Compared with the vehicle-treated group, a significant increase of the NF-L protein level was observed on days 7 (p < 0.01, independent samples t-test; Fig. 4), 14 (p < 0.01, independent samples t-test; Fig. 4) and 28 (p < 0.05, independent samples t-test; Fig. 4) after ONC. It is widely accepted that neuronal loss following nerve injury is greater than what might be expected from the severity of the injury and this is attributable to a variety of processes leading collectively to secondary degeneration [2,4,7,10,13]. Therefore, the term
neuroprotection now commonly refers to the protection of neurons which, following acute insult, do not sustain direct injury, but are adjacent to or surrounded by a damaged milieu and will consequently undergo “secondary degeneration” unless adequately treated [2,4,7,10,13]. In the rat ONC model, there is both direct and indirect damage of the neurones. While direct injury was considered to be the cause of massive cell loss soon after acute injury, indirect toxicity was believed to be responsible for sequential cell death [4,8,13] The loss of RGCs in the first week after the optic nerve injury is mainly due to primary (or direct) damage, and that in week 2 through week 4 after the injury is mainly caused by secondary (or indirect) degeneration [8,13]. In our study, we performed retrograde labelling of RGCs to quantify the number of RGCs at different times following ONC (on days 7, 14 and 28) and conducted immunohistochemistry and Western blot analyses to measure the expression of NF-L proteins in the axons of RGCs. While retrograde labelling of RGCs offers an objective measurement of RGCs survival, the expression of NF-L protein, one subunit of neurofilaments (NFs), is a viable measure of RGC viability [5]. The findings of our study demonstrate that EGCG could increase both the population of survived RGCs and the
Fig. 3. Immunohistochemistry of NF-L in the optic nerve. The optic nerve slices (3 m) were cut in longitudinal sections, then immunostained with rabbit anti-NF-L and restained with haematoxylin to show the distribution of NF-L (dark yellow) and the cell nucleus (blue), respectively. (A) Normal control; (B) negative control; (C) day 7 after ONC with vehicle; (D) day 7 after ONC with EGCG; (E) day 14 after ONC with vehicle; (F) day 14 after ONC with EGCG; (G) day 28 after ONC with vehicle; (H) day 28 after ONC with EGCG. Scale bars indicate 50 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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EGCG prevented secondary degeneration of RGCs. Therefore, how the multi-functions of RGC protection by EGCG play out would be a worthy direction in further investigations. Future research should also look into the protective effects of EGCG beyond week 4 after ONC. Acknowledgments This work was supported by the National Natural Science Foundation of China (grant no. 30973268). The authors thank Professor Timothy Lai at The Chinese University of Hong Kong for advice and critical comments. References
Fig. 4. Western blot analysis of NF-L (A) protein levels relative to actin of the optic nerves in the non-crush group (normal control) and days 7, 14 and 28 after ONC with vehicle (vehicle) or EGCG (EGCG), respectively. Quantitative analysis indicates a significant increment in NF-L expression with EGCG treatment on days 7, 14 and 28 after ONC (B). n = 3 in each case, *p < 0.05, **p < 0.01.
expression of NF-L in optic nerve on days 7, 14 and 28 after ONC. These results suggest that EGCG is capable of reducing both primary and secondary degeneration of RGCs, indicating that EGCG might be a treatment option for acute or chronic optic nerve diseases. Our study also demonstrated that EGCG may protect RGCs even in a small dose. Previous in vivo studies suggested that EGCG at the dose level of 50 mg/kg/day injected intraperitoneally or daily oral administration of 200 ml 0.5% EGCG is capable of protecting RGCs and photoreceptor cells [1,14,15]. Building on recent research on EGCG’ impact on cerebral ischemic damage [6] and aging-associated oxidative damage in rat brain [11], our study confirmed that EGCG is effective of neuroprotection even in a dose as low as 25 mg/kg/day injected intraperitoneally and 2 mg/kg/day given orally. EGCG has been proved to have a wide variety of properties, including antioxidant and free radical scavenging, antiinflammatory, and attenuating glutamate-induced cytotoxicity [12]. Secondary degeneration of RGCs involves mechanisms such as glutamate-induced excitotoxicity leading to calcium overload and oxidative stress, free radical formation, mitochondrial dysfunction and energy failure, enzymatic degradation, membrane instability and resultant inflammation [3]. Our study is limited in the sense that it could not reveal the exact mechanisms in which
[1] B.L.d.S.A.d. Costa, R. Fawcett, G.-Y. Li, R. Safa, N.N. Osborne, Orally administered epigallocatechin gallate attenuates light-induced photoreceptor damage, Brain Res. Bull. 76 (2008) 412–423. [2] A. Faden, Pharmacological treatment of central nervous system trauma, Pharmacol. Toxicol. 18 (1996) 12–17. [3] M. Fitzgerald, C.A. Bartlett, L. Evill, J. Rodger, A.R. Harvey, S.A. Dunlop, Secondary degeneration of the optic nerve following partial transection: the benefits of lomerizine, Exp. Neurol. 216 (2009) 219–230. [4] T.V. Johnson, S.I. Tomarev, Rodent models of glaucoma, Brain Res. Bull. 81 (2010) 349–358. [5] M.K. Lee, D.W. Cleveland, Neurofilament function and dysfunction: involvement in axonal growth and neuronal disease, Curr. Opin. Cell Biol. 6 (1994) 34–40. [6] S. Lee, S. Suh, S. Kim, Protective effects of the green tea (−)-epigallocatechin gallate against hippocampal neuronal damage after transient global ischemia in gerbils, Neurosci. Lett. 28 (2000) 191–194. [7] H. Levkovitch-Verbin, R. Dardik, S. Vander, S. Melamed, Mechanism of retinal ganglion cells death in secondary degeneration of the optic nerve, Exp. Eye Res. (2009) (available online 29.11.09). [8] H. Levkovitch-Verbin, H.A. Quigley, K.R. Martin, D.J. Zack, M.E. Pease, D.F. Valenta, A model to study differences between primary and secondary degeneration of retinal ganglion cells in rats by partial optic nerve transaction, Invest. Ophthalmol. Vis. Sci. 44 (2003) 3388–3393. [9] P. Peng, M. Ko, C. Chen, Epigallocatechin-3-gallate reduces retinal ischemia/reperfusion injury by attenuating neuronal nitric oxide synthase expression and activity, Exp. Eye Res. 86 (2008) 637–646. [10] D.H. Smith, K. Casey, T.K. McIntosh, Pharmacologic therapy for traumatic brain injury: experimental approaches, New Horiz. 3 (1995) 562–572. [11] R. Srividhya, V. Jyothilakshmi, K. Arulmathi, V. Senthilkumaran, P. Kalaiselvi, Attenuation of senescence-induced oxidative exacerbations in aged rat brain by (−)-epigallocatechin-3-gallate, Int. J. Dev. Neurosci. 26 (2008) 217–223. [12] B.A. Sutherland, R.M.A. Rahman, I. Appleton, Mechanisms of action of green tea catechins, with a focus on ischemia-induced neurodegeneration, J. Nutr. Biochem. 17 (2006) 291–306. [13] E Yoles, M. Schwartz, Degeneration of spared axons following partial white matter lesion: implications for optic nerve neuropathies, Exp. Neurol. 153 (1998) 1–7. [14] B. Zhang, D. Rusciano, N.N. Osborne, Orally administered epigallocatechin gallate attenuates retina neuronal death in vivo and light-induced apoptosis in vitro, Brain Res. 1198 (2008) 141–152. [15] 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.