Green Tea Extract Ameliorates Ischemia-Induced Retinal Ganglion Cell Degeneration in Rats

Yaping Yang, Ciyan Xu, Yuhong Chen, Jia-Jian Liang, Yanxuan Xu, Shao-Lang Chen, Shaofen Huang, Qichen Yang, Ling-Ping Cen, Chi Pui Pang, Xing-Huai Sun, Tsz Kin Ng, Yaping Yang, Ciyan Xu, Yuhong Chen, Jia-Jian Liang, Yanxuan Xu, Shao-Lang Chen, Shaofen Huang, Qichen Yang, Ling-Ping Cen, Chi Pui Pang, Xing-Huai Sun, Tsz Kin Ng

Abstract

Purpose: Oxidative stress induced by reduced blood circulation is a critical pathological damage to retinal ganglion cells (RGCs) in glaucoma. We previously showed that green tea extract (GTE) and its catechin constituents alleviate sodium iodate-induced retinal degeneration in rats. Here, we investigated the therapeutic effect of GTE on ischemia-induced RGC degeneration in rats.

Methods: RGC degeneration was induced by ischemic reperfusion in adult Fischer F344 rats. Green tea extract (Theaphenon E) was intragastrically administered 4 times within 48 hours after ischemia. RGC survival, pupillary light reflex, expressions of cell apoptosis, oxidative stress, and inflammation-related proteins were studied.

Results: Ischemic reperfusion significantly induced apoptotic RGCs, RGC loss, and larger constricted pupil area compared to the untreated normal rats. Expressions of activated caspase-3 and caspase-8, Sod2, and inflammation-related proteins as well as p38 phosphorylation were significantly upregulated in the ischemia-injured rats. Compared to the saline-fed ischemic rats, significantly higher number of surviving RGCs, less apoptotic RGCs, and smaller constricted pupil area were observed in the GTE-fed ischemic rats. GTE also reduced the increased protein expressions caused by ischemic injury but enhanced the Jak phosphorylation in the retina. Notably, green tea extract did not affect the survival of RGCs in the uninjured normal rats.

Conclusions: In summary, GTE offers neuroprotection to RGCs under ischemic challenge, suggesting a potential therapeutic strategy for glaucoma and optic neuropathies.

Figures

Figure 1
Figure 1
Schematic diagram of ischemic reperfusion, green tea extract treatments, and experimental assessments in rats. The rats were anesthetized, and ischemic reperfusion injury was induced by the infusion of sterile saline for 2 hours with IOP elevation to 75 mmHg. The rats were intragastrically fed 4 times with 275 mg/kg green tea extract (GTE) at 2, 6, 30, and 46 hours after ischemic reperfusion injury. Protein expression was evaluated on day 2. Retinal ganglion cell (RGC) survival and function were determined on day 14 after ischemic reperfusion injury.
Figure 2
Figure 2
Retinal ganglion cell survival analysis for the green tea extract treatment effect on normal and ischemia-injured rats. Retinal ganglion cell (RGC) survival was evaluated by the immunofluorescence analysis of retinal wholemount for βIII-tubulin on day 14 after ischemic reperfusion (IR) injury. (a) Surviving RGCs in the saline-fed normal rats. (b) Surviving RGCs in the green tea extract- (GTE-) fed normal rats. (c) Surviving RGCs in the saline-fed rats with ischemic injury. (d) Surviving RGCs in the GTE-fed rats with ischemic injury. (e) Quantitative analysis of surviving RGCs in the retina. Data was presented as mean ± standard deviation. Scale bar: 100 μm.
Figure 3
Figure 3
Pupillary light reflex analysis for the green tea extract treatment effect on normal and ischemia-injured rats. Retinal ganglion cell (RGC) function was evaluated by the pupillary light reflex on day 14 after ischemic reperfusion (IR) injury. Pupillary light reflex was measured by the pupil constriction. (a) Surviving RGCs in the saline-fed normal rats. (b) Surviving RGCs in the green tea extract- (GTE-) fed normal rats. (c) Surviving RGCs in the saline-fed rats with ischemic injury. (d) Surviving RGCs in the GTE-fed rats with ischemic injury. (e) Quantitative analysis of the constricted pupil diameter. (f) Quantitative analysis of the constricted pupil area. Data was presented as mean ± standard deviation. Scale bar: 2 mm.
Figure 4
Figure 4
The expression analysis of cell apoptosis, oxidative stress, and inflammation-related proteins in the retina of ischemia-injured rats with green tea extract treatment. (a) Protein expression was evaluated by the immunoblotting analysis on day 2 after ischemic reperfusion injury. Relative fold change analyses on (b) caspase-8 (Casp8), (c) toll-like receptor 4 (TLR4), (d) interleukin-1β (Il-1β), (e) tumor necrosis factor-α (TNF-α), (f) phospho-p38 (p-p38), and (g) phospho-Jak (p-Jak). β-Actin was used as housekeeping protein for normalization. Data was presented as mean ± standard deviation. Compared to the saline-fed normal rats: ∗∗p < 0.01, ∗∗∗p < 0.001. Compared to the saline-fed ischemia-injured rats: #p < 0.05, ##p < 0.01, ###p < 0.001.
Figure 5
Figure 5
Apoptosis analysis of retinal ganglion cells after the green tea extract treatment effect on normal and ischemia-injured rats. Retinal ganglion cell (RGC) apoptosis was evaluated by the TUNEL analysis on retinal sections on day 7 after ischemic reperfusion (IR) injury in (a) the saline-fed normal rats, (b) green tea extract- (GTE-) fed normal rats, (c) saline-fed rats with ischemic injury, and (d) GTE-fed rats with ischemic injury. ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer (on the white-dotted line); green: TUNEL (apoptotic cells); blue: DAPI (nucleus). (e) Quantitative analysis of TUNEL-positive cells in the GCL. Data was presented as mean ± standard deviation. Scale bar: 50 μm.

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