Assessment of neuroprotection in the retina with DARC.

Currently, assessment of new drug efficacy in glaucoma relies on conventional perimetry to monitor visual field changes. However, visual field defects cannot be detected until 20-40% of retinal ganglion cells (RGCs), the key cells implicated in the development of irreversible blindness in glaucoma, have been lost. We have recently developed a new, noninvasive real-time imaging technology, which is named DARC (detection of apoptosing retinal cells), to visualize single RGC undergoing apoptosis, the earliest sign of glaucoma. Utilizing fluorescently labeled annexin 5 and confocal laser scanning ophthalmoscopy, DARC enables evaluation of treatment effectiveness by monitoring RGC apoptosis in the same living eye over time. Using DARC, we have assessed different neuroprotective therapies in glaucoma-related animal models and demonstrated DARC to be a useful tool in screening neuroprotective strategies. DARC will potentially provide a meaningful clinical end point that is based on the direct assessment of the RGC death process, not only being useful in assessing treatment efficacy, but also leading to the early identification of patients with glaucoma. Clinical trials of DARC in glaucoma patients are due to start in 2008.

Introduction

Glaucoma is the major cause of irreversible blindness worldwide and visual loss is attributed to retinal ganglion cell (RGC) death — a hallmark of glaucoma. Glaucoma is commonly linked with raised intraocular pressure (IOP), which has previously been implicated as a major cause of RGC death. Lowering IOP is currently the only clinical therapy available for glaucoma treatment, with an estimated cost of $5 billion annually in America alone by 2011 (Lee et al., 2006). However, pressure-lowering strategies have been shown to be inadequate in the prevention of progressive glaucomatous damage (Collaborative Normal-Tension Glaucoma Study Group, 1998; Oliver et al., 2002). This has provoked much research in non-IOP-lowering strategies, i.e., neuroprotective approaches in glaucoma management.

Currently, the most widely advocated neuroprotective agents in the prevention of RGC death are modifiers of the glutamate pathway, as this, somewhat controversially, is implicated in the development of glaucomatous RGC death (Dreyer et al., 1996; Lipton, 2004b; Guo et al., 2006). Memantine, an NMDA antagonist that has FDA approval for the treatment of Alzheimer's disease (AD), is currently the only neuroprotective drug in clinical trial (phase IV) in glaucoma patients (Hare et al., 2004a, b; Greenfield et al., 2005; Yucel et al., 2006). With the increasing association of glaucoma as a neurodegenerative disorder, there is a trend to use existing neuroprotective strategies that have been shown effective in central nerve system (CNS) diseases (Lipton, 2001). Indeed our recent study has revealed that the amyloid-β drugs used in the treatment of AD patients are also effective in prevention of RGC death in experimental glaucoma (Guo et al., 2007).

At present, monitoring new drug efficacy in glaucoma patients relies on the detection of visual field changes, which has accounted for a long period of follow-up (5 years) necessary for a clinical trial, such as the ongoing memantine clinical trial. Additionally, visual field testing is not a sensitive and accurate method to detect glaucomatous damage as it has been estimated that up to 20–40% of RGCs are lost before visual field defects are detected (Kerrigan-Baumrind et al., 2000). Moreover, given that RGC loss plays a key role in glaucoma with RGC apoptosis being recognized as early event (Quigley et al., 1995; Cordeiro et al., 2004), it would be a fundamental advance if RGC apoptosis could be monitored in evaluating therapeutic efficiency. There is currently no established clinical end point for the assessment of neuroprotective strategies in glaucoma.

We have recently devised an imaging technology called DARC (detection of apoptosing retinal cells) to detect RGC apoptosis in vivo. In this review, we will be focusing on assessment of neuroprotective strategies in the prevention of glaucomatous RGC apoptosis with DARC. DARC will be clinically trialed in 2008, with a view to assessing its role in glaucoma management and neuroprotective drug monitoring.

DARC

Introducing the DARC technique

We have recently established a novel, noninvasive real-time imaging technology to visualize individual RGC apoptosis in vivo in glaucoma-related experimental models. This method, which we have given the acronym DARC (detection of apoptosing retinal cells), employs flurorescently labeled annexin 5 and confocal laser scanning ophthalmoscopy. For the first time, we have been able to image changes occurring in RGC apoptosis over hours, days, and months in vivo (Cordeiro et al., 2004). This technique has recently been demonstrated to be a useful tool in screening neuroprotective strategies in glaucoma-related rat models (Guo et al., 2006, 2007).

Annexin 5-labeled apoptosis and ophthalmoloscopy

Apoptosis plays an important role in both physiology and pathology, particularly glaucoma, where vision loss is attributed to RGC apoptosis and death (Quigley et al., 1995; Kerrigan et al., 1997). Apoptosis is a regulated process of cell suicide, so called programmed cell death (PCD), characterized by cell shrinkage, DNA fragmentation, and chromatin condensation with a lack of inflammatory responses.

The idea of using annexin 5 to detect apoptosis was derived from a cellular phenomenon, first described by Fadok et al. in 1992. They observed that phosphatidylserine (PS), presenting in the inner leaflet of the plasma membrane, becomes exposed on the surface of apoptotic cells, and acts as an “eat me” signal for phagocytes (Fadok et al., 1992). It was further found that annexin 5, a human protein, is able to bind to PS selectively with a high affinity in the presence of Ca2+ (Koopman et al., 1994). As the externalization of PS occurs early in the apoptotic process, well before DNA fragmentation and nuclear condensation can be detected microscopically, annexin 5 binds to the exposed PS thereby identifying early stages of apoptosis.

Because of its properties, annexin 5 has been used in the detection of cells undergoing apoptosis using fluorescein isothiocyanate-labeled annexin 5 (FITC-annexin 5) (Koopman et al., 1994; Dumont et al., 2001). This annexin 5 affinity assay was further developed by labeling annexin 5 with biotin or with several radionuclides to facilitate various protocols for measuring apoptosis both in vitro and in vivo animal models (Blankenberg et al., 1998; Vriens et al., 1998; Dumont et al., 2000; Post et al., 2002; Kenis et al., 2004; Murakami et al., 2004). The availability of 99Tc-labeled annexin 5 produced under good manufacturing practice (GMP) regulations led to the first studies of noninvasive detection of apoptosis in patients (Hofstra et al., 2001; Narula et al., 2001; Boersma et al., 2003; van de Wiele et al., 2003; Kietselaer et al., 2004). Annexin 5 imaging has been demonstrated to be clinically feasible and safe in patients. However, these techniques have never been used in the eye.

Using a related but nonradioactive approach, we have developed an imaging technology that uses fluorescently labeled annexin 5 with direct visualization of the retina to detect the dynamic process of individual cells undergoing apoptosis (Cordeiro et al., 2004). This is possible because the presence of clear optical media in the eye, compared to other organs in the body, allows direct visualization of labeled disease processes as they occur in the eye. The technique uses an imaging device such as a confocal scanning laser ophthalmoscope (cSLO) with an argon laser of 488 nm necessary to excite the administered annexin 5-bound fluorophore, and a photodetector system with a cutoff 521 nm filter to detect the fluorescent emitted light. We have to date used cSLOs with specialized imaging software to compensate for eye movements and improve the signal to noise ratio (von Ruckmann et al., 1995; Wade and Fitzke, 1998).

For imaging, animals under anesthesia are held in a stereotaxic frame, and their pupils dilated. Videos of scanned retinal areas are recorded and assessed for fluorescence using a method we have previously described (Fitzke, 2000). For each eye, a retinal montage is constructed from images captured at the same time point. It is then possible to count a total number of apoptosing RGCs for each time point in vivo (Maass et al., 2007) (Fig. 1).

Fig. 1

DARC in vivo imaging shows retinal ganglion cell (RGC) apoptosis on a retina montage of an OHT rat at 3 weeks after IOP elevation. The white spots represent apoptotic RGCs.

Detection of RGC apoptosis in glaucoma-related animal models with DARC

To evaluate DARC's sensitivity and accuracy in monitoring RGC apoptosis, we have assessed its performance in vivo using several different glaucoma-related animal models and shown that DARC enables the real-time imaging of apoptotic changes occurring in RGCs longitudinally with histological validations.

In a well-established rat model of chronic ocular hypertension (OHT) (Cordeiro et al., 2004; Guo et al., 2005a, b, 2006, 2007), using DARC we revealed that RGC apoptosis occurs in vivo, accounting for 1, 15, 13, 7, and 2% of total RGCs, with RGC losses of 17, 22, 36, 45, and 60% of the original population at 2, 3, 4, 8, and 16 weeks, respectively, after surgical elevation of IOP. In an optic nerve transaction rat model, we recorded RGC apoptosis in 0.3, 1, 8, and 4% of total RGCs, with RGC losses of 0, 3, 49, and 76% at 0, 3, 7, and 12 days, respectively. DARC imaging is also applicable to the mouse eye (Maass et al., 2007). Our results were obtained from a large cohort of animals — with minimal intra- and intervariability reinforcing the reproducibility and repeatability of the technique (Cordeiro et al., 2004; Guo et al., 2005a, b, 2006, 2007).

Additionally, we have developed a model of drug-induced RGC apoptosis using intravitreal staurosporine (SSP), a nonselective protein kinase inhibitor and a well-known potent inducer of neuronal apoptosis (Koh et al., 1995; Belmokhtar et al., 2001; Thuret et al., 2003). Assessment of the SSP model with DARC has shown that it generates rapid and extensive RGC apoptosis with peak levels visualized at 2 h in the rat, and is a useful tool in the assessment of neuroprotective strategies, with strong data attainable within a relatively short time (Cordeiro et al., 2004; Guo et al., 2006).

Assessment of glutamate modulation with DARC

Glutamate at synaptic endings

Glutamate is a predominant excitatory amino acid and important neurotransmitter in the CNS and the retina, involved in a variety of physiological processes and pathophysiological states. During synaptic activity, glutamate, triggered by nerve impulses is released from the presynaptic cell into the synaptic cleft and subsequently binds to glutamate receptors on the postsynaptic membrane to start intracellular signaling cascades. To allow for efficient signaling to occur, glutamate levels in the synaptic cleft have to be maintained at very low levels. This process is regulated by glutamate transporters, which rapidly remove excess glutamate from the extracellular space via a sodium–potassium coupled uptake mechanism.

High levels of extracellular glutamate stimulates glutamate receptors, allowing excessive Ca2+ influx into the cells triggering a multitude of intracellular events in the postsynaptic neuron, which ultimately results in neuronal cell death. This phenomenon is known as excitotoxicity, which has been implicated as an important mechanism of a number of neurodegenerative diseases, such as Alzheimer's (AD), Parkinson's (PD), and Huntington's (HD) diseases.

Glutamate excitotoxicity in glaucoma

There has been increasing evidence that glutamate excitotoxicity has been involved in RGC death in glaucoma although its exact role is controversial. Glutamate-related RGC death was first reported in the late 1950s when Lucas and Newhouse (1957) found that subcutaneous injection of glutamate selectively damaged the inner layer of the retina. A decade ago, Dreyer et al. (1996) reported an increase of glutamate concentration in the vitreous in glaucoma patients and experimental glaucoma monkeys and this finding was further supported by studies using dog (Brooks et al., 1997) and quail (Dkhissi et al., 1999). Although these results have been recently challenged by other researchers (Carter-Dawson et al., 2002; Levkovitch-Verbin et al., 2002; Honkanen et al., 2003; Wamsley et al., 2005), this does not exclude an excitotoxicity mechanism in glaucoma as there are the inherent difficulties in measuring in vivo glutamate levels (Salt and Cordeiro, 2006).

In addition to the possibility of increased release of glutamate from damaged RGCs in glaucoma, a reduced clearance of glutamate may also occur as a result of inefficient uptake by glutamate transporters present in the membranes of neurons and glial cells. In support of this, a significant reduction of the glutamate transporters GLAST (EAAT1) and GLT-1 (EAAT2) has been observed in experimental rat glaucoma retina (Martin et al., 2002). However, instead of a reduction, an increased expression of GLAST in the retinal Muller cells was reported using a similar model (Woldemussie et al., 2004), where the authors suggested a compensatory mechanism. Although other studies have failed to demonstrate a glaucoma-induced defect in retinal glutamate clearance mechanisms (Hartwick et al., 2005), a specific glutamate transporter GLT-1c, which is normally only expressed by photoreceptors, has been identified in RGCs in experimental glaucoma, and it may be indicative of an anomaly in glutamate homeostasis (Sullivan et al., 2006).

Perhaps the most effective exploration of the role of glutamate excitotoxicity in glaucoma is to examine the effect of blocking RGC glutamate receptors. This may be done via ion channel-associated (ionotropic) or G protein-coupled (metabotropic) receptors in the CNS and the retina. RGCs express multiple subtypes of these receptors (Hamassaki-Britto et al., 1993; Brandstatter et al., 1994; Hartveit et al., 1995; Fletcher et al., 2000). The ionotropic (iGlu) receptors include N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and KA (kainite) subtypes; and among them, NMDA receptors, which are the most permeable to Ca2+, have been extensively studied.

Postsynaptic NMDA receptors are heteromeric ion channel complexes that consist of two NR1 and two NR2 subunits that can be either of the NR2A, -2B, -2C, or -2D type. The NR1 contains a specific glutamate-binding site, whereas the NR2 binds to glycine, which acts as a co-agonist for receptor activation. When both glutamate and glycine bind onto the receptors, the cells are depolarized, resulting in the release of Mg2+, which blocks the channel. The opening of the channel is transient under normal physiological condition, allowing appropriate amounts of extracellular Ca2+ and Na+ to flood into the cell to initiate intracellular signaling cascades. However, overexpression of extracellular glutamate causes the channel to stay open longer than needed; as a result, excessive Ca2+ flows into the cell triggering cell death.

Blocking NMDA receptors by specific antagonists has been shown to be effective in reducing RGC death in experimental glaucoma. Memantine, an uncompetitive NMDA antagonist, is perhaps the best-known glutamate modifier and has recently been shown to be clinically useful in the treatment of moderate to severe AD (Lleo et al., 2006). Memantine has been demonstrated to be a highly effective neuroprotective agent in various animal models of RGC death (Lagreze et al., 1998; Osborne, 1999; Kim et al., 2002; WoldeMussie et al., 2002). Application of memantine significantly increased RGC survival in experimental rat glaucoma (WoldeMussie et al., 2002), and a similar result was reported using the DBA/2J transgenic mouse model of glaucoma (Schuettauf et al., 2002). Additionally, memantine has shown to be preventive of not only structural damage but also functional loss in experimental monkey glaucoma (Hare et al., 2004a, b). Memantine is currently in a phase IV clinical trial of glaucoma, the results of which are eagerly awaited.

Assessment of glutamate modulation in glaucoma-related models with DARC

Using DARC, we have assessed the effects of different glutamate modulation strategies, including a nonselective (MK801) and a selective (ifenprodil) NMDA receptor antagonist and a metabotropic glutamate receptor agonist (mGluR Group II, LY354740), in glaucoma-related rat models in vivo. We have shown that DARC is a useful tool in screening neuroprotective therapies and monitoring RGC apoptosis in experimental glaucoma (Guo et al., 2006).

Like memantine, MK801 is a broadspectrum NMDA antagonist and has been reported to protect retina neurons from NMDA- and pressure-induced toxicity (el-Asrar et al., 1992; Chaudhary et al., 1998). Ifenprodil, an NR2B subunit-selective NMDA antagonist, is specific against SSP-induced cell death (Williams et al., 2002). LY354740 is a highly potent and selective group II metabotropic receptor (mGluR) agonist, which has been documented to be neuroprotective against NMDA- or SSP-induced neuronal death in rat CNS (Monn et al., 1997; Bond et al., 1998; Kingston et al., 1999). Metabotropic receptors have been classified into three groups. There is evidence that activation of group I mGluRs increases neuronal excitation, whereas that of group II and III mGluRs reduces synaptic transmission; therefore, group II and III mGluR agonists and group I antagonists can be thought to be neuroprotective (Nicoletti et al., 1996). All mGluRs have been found to be expressed in the retina (Hartveit et al., 1995; Shen and Slaughter, 1998; Tehrani et al., 2000; Robbins et al., 2003).

In this study (Guo et al., 2006), we first assessed the effects of each single agent on RGC apoptosis in our SSP model. Using DARC, we showed that all treatments reduced RGC apoptosis in a dose-dependent manner, and that MK801 was more effective than ifenprodil. However, MK801 has been reported to be neurotoxic due to its high affinity for the NMDA receptor channel, causing its accumulation in the channels and blocking critical normal functions (Lipton, 1993, 2004a). To minimize its toxicity but still take advantage of the neuroprotective properties, we also looked at the effect of combining low-dose MK801 with LY354740 and found this combination to be most effective in preventing RGC apoptosis compared to either agent alone, which may be attributed to their different pharmacological properties in modulating glutamate excitatory transmission.

We then applied the most optimal combination regimens of MK801 and LY354740 to an OHT model at different time points (0, 1, and 2 weeks after IOP elevation), and our DARC results revealed that the most effective timing of the treatment application was at 0 weeks (the time of IOP elevation) (p<0.05, Fig. 2). We believe this is due to the application of glutamate modulators at the time of the primary insult (IOP elevation) leading to maximal inhibition of glutamate release from primary injured RGCs, resulting in the prevention of secondary degeneration — a process in which RGCs that survive the initial injury are subsequently damaged by the toxic effects of the primary degenerating neurons (Levkovitch-Verbin et al., 2001, 2003; Kaushik et al., 2003). Our DARC data strongly supports the involvement of glutamate in glaucoma (Guo et al., 2006; Salt and Cordeiro, 2006).

Fig. 2

DARC reveals combination effects of glutamate modulators on reduction of RGC apoptosis in an OHT model. Pressure-induced RGC apoptosis at 3 weeks (A) was significantly reduced by combination treatment of MK801 and LY354740 (B). Timing administration showed that the most effective treatment application was at 0 weeks (the time of IOP elevation, C).

Assessment of targeting the amyloid-β pathway with DARC

Amyloid-β neurotoxicity in AD

The Alzheimer protein amyloid-β (Aβ) is the major constituent of amyloid plaques in AD. Aβ is an amino acid peptide derived from the proteolytic cleavage of amyloid precursor protein (APP), a transmembrane protein. There are two catabolic pathways being identified for APP processing: the nonamyloidogenic pathway relying on α-secretase activity and resulting in secretion of soluble forms of APP, functioning as a cell surface signaling molecule to modulate neurite outgrowth, synaptogenesis and cell survival (Li et al., 1997; Pastorino and Lu, 2006), and the amyloidogenic pathway where β- and γ-secretase activities lead to Aβ generation. Aggregation of Aβ in the brain is believed to be the primary driver of neurodegeneration and cognitive decline leading to dementia (Auld et al., 2002), and Aβ neurotoxicity has been involved in AD's neuropathology, although the proximate cause of the neurodegeneraton responsible for cognitive impairment is not clear (Hardy and Selkoe, 2002; Pepys, 2006). Blocking the APP amyloidogenic pathway has been shown to be a promising approach to prevent neuronal damage in AD.

Amyloid-β implication in glaucoma

Amyloid-β has recently been reported to be implicated in the development of RGC apoptosis in glaucoma, showing caspase-3-mediated abnormal processing of APP with increased expression of Aβ in RGCs in the OHT rat (McKinnon et al., 2002) and DBA/2J transgenic mice (Goldblum et al., 2007). In addition, decreased vitreous levels of Aβ have been found in patients with glaucoma compared to controls, suggesting the deposition of Aβ in the retinal (Yoneda et al., 2005). Further evidence of a link between glaucoma and AD has emerged from studies showing that patients with AD have RGC loss and reduced thickness of the retinal nerve fiber layer (RNFL) associated with typical glaucomatous changes, such as optic neuropathy and visual functional impairment (Blanks et al., 1996a, b; Parisi et al., 2001; Danesh-Meyer et al., 2006; Iseri et al., 2006; Tamura et al., 2006; Berisha et al., 2007), as is also the case in PD (Bayer et al., 2002). This indicates similar pathological mechanisms involving Aβ leading to RGC loss as implicated in the brain (Loffler et al., 1995; Vickers et al., 1995; Archer et al., 1998; Johnson et al., 2002).

Using DARC, we have recently provided further evidence from experimental glaucoma supporting the involvement of Aβ in development of glaucomatous RGC apoptosis (Guo et al., 2007). We observed a significant increase of Aβ deposition in the RGC layer in OHT model compared to control, with Aβ colocalized to apoptotic RGCs. DARC imaging data further showed that application of exogenous Aβ peptide induced significant RGC apoptosis in vivo in a dose- and time-dependent manner.

Assessment of targeting Aβ pathway in experimental glaucoma with DARC

The finding of increased Aβ deposition in experimental glaucoma and its induction of RGC apoptosis in vivo suggests that Aβ could be a factor mediating the apoptotic changes in RGC cells in our glaucoma model. To examine our hypothesis that targeting Aβ formation and aggregation might reduce RGC apoptosis, we examined three different agents, including a β-secretase inhibitor (βSI), an anti-Aβ antibody (Aβab), and Congo red (CR) (Guo et al., 2007) (Fig. 3).

Fig. 3

The amyloid-beta (Aβ) pathway and its targeting. Abnormal processing of APP causes the generation of Aβ, leading to neuronal death. Using DARC, we assessed the effects of targeting Aβ formation and aggregation on prevention of glaucomatous RGC apoptosis by a β-secretase inhibitor (βSI), an anti-Aβ antibody (Aβab), and Congo red (CR).

β-Secretase, a membrane-anchored aspartic protease, is responsible for the initial step of APP cleavage in the amyloidogenic pathway leading to the generation of Aβ; βSIs therefore inhibit the generation of Aβ, with evidence of in vitro and in vivo efficacy in AD-related models (Citron, 2002; Yamamoto et al., 2004). CR, a dye commonly used to stain amyloid-β histologically, has been shown to completely block Aβ aggregation and toxicity in rat hippocampal neuron cultures (Lorenzo and Yankner, 1994), and it is believed that its inhibitory effects are the result of an interference with Aβ protein misfolding, fibril formation (Lorenzo and Yankner, 1994; Hirakura et al., 1999). Aβabs are thought to work by not only blocking Aβ aggregation (Bard et al., 2000) but also increasing Aβ clearance in AD-related animal models (Vasilevko and Cribbs, 2006) (Fig. 3).

Our DARC data showed that all three treatments altered the profile of RGC apoptosis in a temporal manner by delaying the development of peak RGC apoptosis as well as reducing the peak level of RGC apoptosis. Among them, the anti-Aβ ab appeared the most effective in the prevention of RGC apoptosis compared with CR and the βSI. The single application of the Aβab showed a prolonged effect up to 16 weeks following IOP elevation. In comparison, CR appeared to have a shorter window of RGC protection and βSI showed no significant effect on glaucomatous RGC apoptosis (Fig. 4).

Fig. 4

DARC in vivo images show the effects of single agents including an Aβ antibody (Aβab, A and B), Congo red (CR, C and D), and a β-secretase inhibitor (βSI, E and F) on RGC apoptosis at 3 (A, C, and E) and 16 (B, D, and F) weeks after IOP elevation. Aβab and CR caused significant percentage reduction of RGC apoptosis at 3 weeks compared to control (G).

Perhaps the most significant finding of the work with DARC has been combination therapy, targeting three different aspects of the Aβ pathway. In combination, the neuroprotective effect of all three agents (triple therapy) was significantly improved at 3 weeks after IOP elevation compared with Aβab alone (Fig. 5). The combination therapy produced the maximal reduction of RGC apoptosis (>80%).

Fig. 5

Effects of combination Aβ-targeting therapy on RGC apoptosis. Compared to nontreatment control (A), DARC imaging shows triple therapy (C, Aβab+CR+βSI) was more effective than Aβab alone (B) in reduction of RGC apoptosis in an OHT model.

Using DARC, we have highlighted that Aβ is a likely mediator of pressure-induced RGC death and that targeting multiple stages in the Aβ pathway may provide a potential neuroprotective strategy in glaucoma management.

Assessment of coenzyme Q10 in glaucoma-related models with DARC

Coenzyme Q10 (CoQ10) is an important insoluble component of the mitochondrial respiratory chain where it transports electrons from complexes I and II to complex III, by which ATP is produced. CoQ10 has been reported to afford neuroprotection, preventing neuronal death (Papucci et al., 2003). CoQ10 acts as a potent antioxidant by maintaining the mitochondrial membrane potential and inhibiting ROS generation when neuronal cells are subject to oxidative stress (McCarthy et al., 2004; Somayajulu et al., 2005). CoQ10 has been shown to prevent RGC apoptosis in a pressure-induced transient ischemia model by minimizing synaptic glutamate increase and inhibiting the MPTP formation (Nucci et al., 2007). CoQ10 has been safely used in patients with neurodegenerative disorders (Levy et al., 2006; Liu, 2007).

We have investigated the effects of topical CoQ10 on reducing RGC apoptosis in vivo using our SSP rat model (Cordeiro et al., 2007). Our DARC imaging results showed that CoQ10 0.1% significantly reduced SSP-induced RGC apoptosis compared to CoQ10 0.05% and carrier. The most effective timing administration was at 1 h after SSP application. The effects of CoQ10 on preventing RGC apoptosis may be attributed to its property of inhibiting mitochondrial depolarization, cytochrome c release, and caspase-9 activation (Papucci et al., 2003).

Summary

Currently, there is no good and quick method for assessing neuroprotection in glaucoma. The only neuroprotective drug that has undergone large scale clinical trial in glaucoma, memantine, has relied on visual field status as a defined end point, which has led to an expensive 6-year period of follow-up — with still no published outcome. We believe our recently developed DARC imaging technology is a major advance in this area, with the potential of providing a much needed new end point in glaucoma. This is supported by our experimental work, which has clearly shown the power of this technology in assessing neuroprotective strategies. Clinical trials of DARC are due to start in 2008, and we eagerly await the results.

Abbreviations

anti-Aβ antibody

Alzheimer's disease

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate

amyloid precursor protein

brain-derived neurotrophic factor

Congo red

coenzyme Q10

confocal scanning laser ophthalmoscopy

detection of apoptosing retinal cells

fluorescein isothiocyanate

glutamate transporter

Huntington's disease

kainite

mitochondria permeability transition pore

N-methyl-d-aspartate

ocular hypertension

Parkinson's disease

phosphatidylserine

retinal ganglion cell

reactive oxygen species

β-secretase inhibitor

staurosporine Aβ: Amyloid-β

tumor necrosis factor-α