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elevated IOP, may adversely affect neuronal survival by inducing cell death (Kitano et al., 1996; Rosenbaum et al., 1997; Gross et al., 1999; Tezel and Wax, 1999, 2000; Osborne et al., 2004). Oxidative stress causes degeneration of trabecular meshwork (Tamm et al., 1996; Izzotti et al., 2003; Sacca` et al., 2005) leading to alterations in the aqueous outflow pathway, ultimately increasing IOP that, in turn, damages RGCs. Free radical species can cause neuronal cell death by inhibition of key enzymes of the tricarboxylic acid cycle, the mitochondrial electron transport chain, and the mitochondrial calcium homeostasis, leading to defective energy metabolism (Patel et al., 1996; Duchen, 2000; Beal, 2005).

Growing evidence also supports the hypothesis that oxidative stress is the leading mechanism of excitotoxic, glutamate-mediated RGC loss in vitro (Luo et al., 2001) and in vivo (Nucci et al., 2005). Accumulation of free radical species may damage glutamate transporter proteins (Muller et al., 1998), decreasing the capacity of neurons and astrocytes to metabolize glutamate, resulting in abnormal synaptic concentration of glutamate and excitotoxicity (Trotti et al., 1996; Sandhu et al., 2003). Moreover, glutamine synthetase is also oxidatively modified in ocular hypertensive eyes (Tezel et al., 2005). In addition, retinal glutamateinduced damage has been shown to be mediated in part through nitric oxide (NO), a highly reactive oxidant (Nucci et al., 2005).

It is well established that free radical scavengers are useful pharmacological tools to improve mitochondrial function and prevent neuronal cell death under excitotoxic conditions (Lipton and Rosenberg, 1994). Free radical scavengers when combined with trophic factors result in increased survival of RGCs in ocular hypertensive eyes (Ko et al., 2000) and in combination with a nitric oxide synthase inhibitor potentiate the neurotrophic effect of brain-derived neurotrophic factor on axotomized RGCs (Klocker et al., 1998).

Coenzyme Q10 (CoQ10) is an important component of the mitochondrial electron transport chain, and it is also a potent antioxidant that has been shown to afford neuroprotection in Huntington’s and other neurodegenerative diseases (Beal and Matthews, 1997; Matthews et al., 1998; Beal,

1999, 2004; Ferrante et al., 2002; Shults et al., 2002, 2004). Neuroprotection by CoQ10 has in part been attributed to its free radical scavenger ability and to a specific inhibition of the mitochondrial permeability transition pore (PTP), a channel whose opening causes the mitochondrial membrane potential collapse that leads to apoptosis (Papucci et al., 2003).

In this chapter, using an established animal model of retinal ischemia/reperfusion in rat (Osborne et al., 2004), we will be focusing on assessment of neuroprotective properties of CoQ10 in RCG damage in rat retina.

Ischemia model

High IOP-induced ischemia is a frequently used animal model for retinal ischemia research (see Osborne et al., 1999, 2003, 2004) that produces pathological features almost identical to those seen in patients after central retinal artery or ophthalmic artery occlusion but may also represent a model of acute-angle closure glaucoma. The latter hypothesis is based on the observation that in animal subjected to ischemia caused by elevated IOP, a retinal damage similar to that seen in human glaucoma is produced, whereas occlusion of retinal blood vessels (e.g., occluding the carotid or vertebral arteries) causes a different pattern of damage. In particular, clamping of the carotids showed that opsin mRNA levels were decreased dramatically, indicating that the photoreceptors were preferentially affected. In contrast, ischemia induced by elevated IOP induces a large decrease in the RGC surface-associated antigen, Thy-1, whereas opsin levels were unaffected, indicating that the ganglion cells were preferentially affected (Osborne et al., 1999).

Neuroprotective effect of Coenzyme Q10 against cell loss yielded by transient ischemia in the RGC layer

Retinal ischemia for 45 min followed by 24 h reperfusion causes retinal damage and cell death (Nucci et al., 2005). Under these experimental conditions, reduction in the number of cells in the

RGC layer is also observed. The loss of RGC seems to occur via an apoptotic mechanism and this event seems to be initiated early after the ischemic insult. In fact, DNA fragmentation, evaluated by the occurrence of positive cells by terminal transferase (TdT) dvDP nick end-labeling (TUNEL) technique (Gavrieli et al., 1992), in the RGC layer was observed as early as 6 h after reperfusion (Nucci et al., 2005). The mechanism underlying cell loss implicates overactivation of N-methyl-D-aspartate (NMDA) and non-NMDA subtypes of glutamate receptors and consequent accumulation of NO (Nucci et al., 2005). Topical treatment with antioxidant agents such as CoQ10 (solution of CoQ10 0.1%+Vit-E 0.5%+saline+EDTA 0.1%) and vitamin E (Vit-E) minimizes retinal damage and cell death (Nucci et al., 2007; Fig. 1).

The mechanism underlying neuroprotection afforded by CoQ10 is not known, although it is conceivable that a free radical scavenging mechanism may play a minor role. In fact, neuroprotection afforded by CoQ10 is far greater than that provided by treatment with Vit-E alone. In addition, further support to our hypothesis comes

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from the neuroprotection afforded by CoQ10 in the course of experimental neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases whose pathogenesis implicates failure of mitochondrial energy metabolism (Beal and Matthews, 1997; Matthews et al., 1998; Beal, 1999, 2004; Ferrante et al., 2002; Shults et al., 2002, 2004; Cleren et al., 2008). Accordingly, an alternative hypothesis would be that CoQ10 reduces the detrimental action of ischemia/reperfusion on mitochondrial energy metabolism and, consequently, on the function of glutamate transporters, thus limiting accumulation of extracellular glutamate and preventing apoptotic death of RGCs. In fact, it is well documented that excessive activation of glutamate receptors via the excitotoxic cascade leads to the PTP formation and cytochrome c, a member of the mitochondrial electron transport chain, to be released from the mitochondrial intermembrane space into the cytosol, where it functions as a pro-apoptotic factor committing the RGC to death (see Kroemer and Reed, 2000). Incidentally, CoQ10 has been shown to inhibit apoptosis by maintaining the PTP in the