protection from ischemia-reperfusion injuries to the retina as a whole and to ganglion cells in particular.
Nicotinamide
Nicotinamide is a precursor of NADH, which is a substrate for complex 1 (NADH-CoQ oxidoreductase). It is also an inhibitor of poly-ADP-ribose polymerase (PARP), an enzyme which is activated by DNA damage and in turn depletes both NADH and ATP (Eliasson et al., 1997). Activation of PARP plays a role in neuronal injury induced by both ischemia and MPTP toxicity (Mandir et al., 1999). Inhibition of PARP has also been shown to
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attenuate neuronal injury and ATP depletion produced by MTPT (Cosi and Marien, 1998). Appropriate nicotinamide pretreatment, in a middle cerebral artery occlusion model of stroke, reduced infarct volume, augumented cerebral blood flow, and increased brain NAD+ levels in ischemic penumbra (Sadanaga-Akiyoshi et al., 2003). Moreover, in human studies, nicotinamide administration improved phosphocreatine/ATP ratios in certain patients. Recent studies on retinal cell cultures and rats support the supposition that appropriate nicotinamide supplementation might benefit glaucoma patients. In one series of experiments, nicotinamide (500 mg/kg) was administered intraperitoneally just before ischemia and into the vitreous (5 ml of a 200 mM solution) immediately
Fig. 6. Protein levels in whole retinas (NF-L, caspase-3, GFAP, and PARP) and optic nerves (NF-L and tubulin) relative to their individual amount of actin of eyes given ischemia where saline (n ¼ 6) or nicotinamide (n ¼ 6) was administered, and compared with the retina and optic nerve of the eye that in each case had not received any treatment (control). Results expressed as mean values7SEMs. It can be seen that significant ( po0.01) changes in all proteins were found in retinas given ischemia (saline) relative to the untreated control retinas. These changes in the retinas given ischemia are significantly blunted ( po0.05) when the animals are treated with nicotinamide.
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after the insult (Ji et al., 2008). Seven days after ischemia (IOP raised to120 mmHg for 50 min), retinas were analyzed for the localization of various antigens as well as the content of certain proteins and mRNAs. In addition, optic nerves were analyzed for their NF-L content. Ischemia/ reperfusion affected a number of parameters in the retina that included a decrease in ganglion cell proteins (Thy-1 and NF-L) and mRNAs (NF-L and melanopsin) (see Fig. 6). Also, ischemia/reperfusion caused clear changes in the localization of nitric oxide synthase and ChAT and an activation of a certain protein/mRNAs (caspase-3, PARP) involved in apoptosis. In addition, ischemia/reperfusion caused a drastic loss of NF-L protein in the optic nerve. All these effects induced by ischemia/reperfusion were significantly blunted by nicotinamide treatment (Fig. 7).
Epigallocatechin gallate
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 (Mitscher et al., 1997; Mandel et al., 2005). 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, increases endogenous antioxidant defenses, counteracts inflamma- tion-mediated neuronal injury, and causes a downregulation of pro-apoptotic genes. 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
Fig. 7. Staining of RGC-5 cells exposed to light for 96 h (B) compared with cells maintained in the dark (A) by the APOPercentageTM methodology for apoptosis (arrows). Inclusion of 0.5 mm nicotinamide (C) or 100 mM NU1025, a known PARP inhibitor (D), clearly reduced the positive staining of cell membranes as an indication for apoptosis induced by light. Scale bar ¼ 20 mm. (See Color Plate 24.7 in color plate section.)
(Thiagarajan et al., 2001; Vinson and Zhang, 2005). Moreover, our recent studies have shown that photoreceptor degeneration induced by an intraocular injection of sodium nitroprusside (nitric oxide generator) can be attenuated by coinjection with EGCG (Zhang and Osborne, 2006) and that a systemic administration of EGCG attenuates retinal damage caused by ischemia/ reperfusion (Zhang et al., 2007). These studies provide 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 (Suganuma et al., 1998) shows orally administered EGCG to reach various tissues including the central nervous system, within 6 h. Moreover, a second administration after a 6 h interval enhances tissue levels four to six times above that of a single administration. 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 (Isbrucker et al., 2006). It has also been estimated that the half-life of green tea catechins 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 (Zhu et al., 2000). 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. Oral administration of EGCG at a high daily dose of 800 mg is known to be safe and without side effects in healthy individuals. Recently, we therefore examined whether orally administrated EGCG to rats can attenuate retinal ischemia (Zhang et al., 2008). Ischemia was delivered to one eye of a number of rats by raising the intraocular pressure, where EGCG (0.5%, each rat intake was approximately 200 ml/day) was present in the drinking water of half the animals three days before ischemia and also during the next five days of reperfusion. The ERGs of both eyes from all rats were recorded before ischemia and five days following ischemia. Seven days after ischemia, either retinas from both eyes of all rats were analyzed for the localization of various
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antigens or extracts were prepared for analysis for the level of specific proteins and mRNAs. Ischemia/reperfusion to the retina affected a number of parameters. These included the localization of Thy-1 and choline acetyltransferase, the a- and b-wave amplitudes of the ERG, the content of certain retinal and optic nerve proteins, and various mRNAs. Significantly, EGCG statistically blunted many of the effects induced by ischemia/ reperfusion (see Figs. 8 and 9). This included ischemia-induced alterations to proteins associated with ganglion cells (NF-L and tubulin), Mu¨ller cells (GFAP), photoreceptors (rhodopsin kinase), and apoptosis (caspase-3, PARP, and AIF).
Fig. 8. NF-L (A) and tubulin (B) proteins relative to actin in the nonischemic optic nerves (control) and following ischemia/ reperfusion with vehicle treatment (vehicle) or EGCG treatment (EGCG). Ischemia/reperfusion caused a significant decrease in the amounts of both NF-L and tubulin proteins. EGCG treatment significantly attenuated this reduction. Data are means with error bars indicating 7SEMs. n ¼ 8 in each case,po0.05, and po0.01.