(Chien et al., 2003), and they have recently been confirmed by additional studies in a rat model of glaucoma (Oltmanns et al., 2008). Porcella et al. (2001) reported that WIN-55-212-2 decreases IOP in patients with a type of glaucoma that was refractory to conventional treatment. eCBs like AEA (Pate et al., 1998) or noladin ether (Laine et al., 2002a) have also been found to reduce IOP without producing systemic toxic effects. In addition, the IOP-lowering effects of AEA have been shown to be potentiated by simultaneous administration of phenylmethylsulfonyl fluoride, a compound that inhibits AEA breakdown (Laine et al., 2002b). The latter finding highlights the potential therapeutic perspectives offered by drug-induced modulation of the endocannabinoid tone in the control of IOP. In a recent pilot study, sublingual administration of THC reduced IOP in patients with glaucoma, without producing any significant systemic side effects (Tomida et al., 2004). It seems clear that, while the use of marijuana for the treatment of glaucoma is not supported by scientific evidence, other molecules — natural and synthetic — that interact with the ocular endocannabinoid system are offering new perspectives for the control of IOP.

The mechanisms underlying the effects of (e)CBs on IOP have yet to be completely defined. Straiker’s demonstration in 1999 of the presence of CB1 receptors in the pigmented epithelium of the ciliary body, the trabecular meshwork, the Schlemm canal, and the ciliary muscle suggested that CB1 receptor agonists might influence both the production and drainage of aqueous humor. This hypothesis was subsequently confirmed by studies in which pretreatment with CB1 receptor antagonists prevented the IOP-lowering effects normally observed with the metabolically stable analog of anandamide, methanandamide (Pate et al., 1997), or with the synthetic CBs CP55,940 (Pate et al., 1998) and WIN-55,212-2 (Song and Slowey, 2000; Hosseini et al., 2006; Oltmanns et al., 2008). In addition, Lograno and Romano (2004) showed that activation of the CB1 receptor by AEA or CP55,940 caused contraction of the ciliary muscle, an event known to promote outflow of the aqueous humor through the trabecular meshwork. In this context, activation of CB1

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receptors present in blood vessels within the ciliary body is thought to reduce the production of aqueous humor by inducing vasodilatation. Chien et al. (2003) confirmed this hypothesis by demonstrating that the pressure-lowering effects of WIN- 55212-2 in monkeys with experimentally induced glaucoma are caused by a reduction of approximately 18% in aqueous humor production, an effect that is mediated by the CB1 receptor.

It has recently been suggested that eCBs might also have IOP-lowering effects that are not receptormediated. Rosch et al. (2006) found that treatment with AEA, its stable analog methanandamide, or with THC increases cyclooxygenase-2 (COX-2) expression in cultured cells from the nonpigmented epithelium of the ciliary body. As a result, the supernatants from these cultures contained higher levels of the COX-2 product prostaglandin E2 (PGE2) and of matrix metalloproteinases-1, -3, and -9. These mediators are known to be involved in remodeling of the aqueous humor outflow pathways, and thus contribute to the regulation of IOP (Weinreb and Lindsay, 2002). This finding was consistent with a report by Maihofner et al. (2001), who showed that COX-2 expression in patients with advanced glaucoma is considerably lower than that observed in healthy individuals (Maihofner et al., 2001). Patients with chronic glaucoma or steroidinduced glaucoma also have lower aqueous humor levels of PGE2 than those of patients undergoing surgery for cataract (Maihofner et al., 2001). Therefore, the eCBs might lower IOP via activation of cyclooxygenases as well as through receptordependent mechanisms. In line with this, studies in animals models of glaucoma have shown that the IOP-lowering effects of these agents are attenuated by drugs that block cyclooxygenases, such as indomethacin and steroids (Pate et al., 1996; Green et al., 2001).

Endocannabinoids and neuroprotection

Experimental findings support the view that drugs capable of interacting with the endocannabinoid system exert specific neuroprotective effects (Van der Stelt and Di Marzo, 2005). These have been reported in experimental models of excitotoxic

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CNS damage, including stroke (Nagayama et al., 1999; Amantea et al., 2007), head trauma (Panikashvili et al., 2001), epilepsy (Marsicano et al., 2003), multiple sclerosis (Pryce et al., 2003; Centonze et al., 2007), and other neurodegenerativeneuroinflammatory diseases (Maccarrone et al., 2007). There is also a growing body of experimental data supporting a role for excitotoxicity in the pathogenesis of glaucomatous neuron injury (Nucci et al., 2005a). Subcutaneous or intravitreal administration of glutamate was first shown to produce toxic effects on retinal cells in the 1950s (Lucas and Newhouse, 1957; Sisk and Kuwabara, 1985). Later studies showed that even slight increases in glutamate levels cause retinal damage (Samy et al., 1994), primarily in the large RGCs (Glovinsky et al., 1993), which are also the first cells to display signs of glaucomarelated injury.

Recently, using a microdialysis technique, we have reported acutely increased concentrations of glutamate in the retina of rat in which high IOPinduced retinal ischemia is accompanied by delayed RGC death (Nucci et al., 2005b). These neurochemical data lend support to similar data obtained from human glaucomatous eyes (Dreyer et al., 1996; but see also Carter-Dawson et al., 2002; Honkanen et al., 2003) and from experimental animals (Adachi et al., 1998; Louzada-Ju´nior et al., 1992; but see also Muller et al., 1997; Kwon et al., 2005). Involvement of the excitotoxic cascade in glaucoma has been confirmed by studies in which glutamate receptor antagonists conferred neuroprotection in in vitro and in vivo models of RGC death (Sucher et al., 1997; Adachi et al., 1998). Under the experimental conditions of excitotoxicity, RGC might occur via apoptosis and this can be prevented by antagonists of both NMDA and non-NMDA glutamate receptors (i.e., MK801 and GIKI52466, respectively) (Nucci et al., 2005b). Similar neuroprotection is afforded by L-NAME, an inhibitor of nitric oxide synthase, or by free-radical scavengers such as coenzyme Q10 and vitamin E (Nucci et al., 2007a). These findings strengthen the hypothesis that excessive accumulation of extracellular glutamate plays a role in glaucoma. Via activation of NMDA and non-NMDA glutamate receptors, this excitatory neurotransmitter increases intracellular

levels of calcium, which activate nitric oxide synthase and lead to the release of free nitrogen radicals with subsequent death of RGCs.

Interestingly, El-Remessy et al. (2003) found that systemic administration of THC or cannabidiol (CBD), a major nonpsychotropic constituent of cannabis, prevents RGC death triggered by intravitreal administration of NMDA in rat, and this effect was associated with reduced formation of peroxynitrites. The neuroprotective effects of these CBs was partially inhibited by SR141716A, a selective CB1 receptor antagonist. These findings are consistent with the recent report by Crandall et al. (2007), who found that 20 weeks of treatment with THC lowers IOP and reduces RGC death by approximately 75% in animals with chronic experimentally induced glaucoma.

As summarized above, the endocannabinoid system present in the human retina includes proteins that synthesize, transport, and hydrolyze anandamide, along with CB1 and TRPV1 receptors that are activated by AEA. Our research group has investigated the role of this endogenous system in the neuronal damage that follows acute ocular hypertension (Nucci et al., 2007b). In our experimented model, retinal ischemia induced by ocular hypertension was associated with a 25% reduction in intraretinal levels of AEA (Fig. 1). This effect seems to be the result of an altered endocannabinoid metabolism in the retina. Indeed, as early as 3 h after the acute hypertonic insult, we demonstrated progressive increases in the expression and activity of FAAH, the enzyme that hydrolyzes anandamide. Six and twelve hours after the insult, FAAH activity displayed increases of 150 and 230%, respectively, over baseline values. In contrast, the increased IOP did not have any significant effect on the activities of other enzymes involved in the metabolism of AEA, such NAPE-PLD, which is mainly responsible for the biosynthesis of AEA, or AMT, which transports this substance across cell membranes (Fig. 1).

Collectively, our study seems to indicate that acute elevation of IOP is associated with a reduction in retinal endocannabinoid tone, secondary to increased degradation of anandamide. To

determine whether these events contributed to RGC death in our model, we pretreated animals with URB597, a selective FAAH inhibitor, and evaluated the retinal damage provoked by the ocular hypertensive insult in terms of total number of cells in the ganglion layer and levels of mRNA for THY-1, a specific marker of RGCs. URB597 pretreatment prevented the increase in FAAH activity triggered by acute ocular hypertension and diminished RGC loss, compared with that observed in untreated controls (Fig. 2, Table 1), suggesting that reduced levels of AEA caused by enhanced FAAH activity do indeed play a role in the retinal cell loss provoked by acute ocular hypertension. This hypothesis is further supported by our observation that intravitreal administration of methanandamide (Fig. 2, Table 1), prevents ganglion-cell death in rats exposed to retinal

ischemia caused by acute ocular hypertension. The neuroprotective effects exerted by methanandamide seem to be related to activation of CB1 and TRPV1 receptors, since they are abolished by treatment with antagonists of these receptors, like SR141716A and capsazepine, respectively (Fig. 2, Table 1). Collectively, our findings seem to indicate that the retinal endo cannabinoid system provides a form of neuroprotection that can be weakened under certain conditions, leading to the activation of cell death cascades. Restoration of physiological levels of anandamide with agents that inhibit its enzymatic degradation or act as CB1 or vanilloid receptor agonists (e.g., methanandamide) appears to be a promising strategy for strengthening the protective effect of endocannabinoids in the retina, and thus for preventing cell loss.

Relative Expression to control Retina (%)

120

100

80

60

40

20

0

THY-1 expression analysis

*

*

**

**

The high IOP-induced retinal ischemia model has been widely used in studies on neuroprotection (Osborne et al., 1999, 2004). Like the observation in chronic glaucoma in humans, the retinal damage in this model is largely confined to the ganglion cells, whereas in models of pure retinal ischemia without ocular hypertension (i.e., those caused by ligation of the ophthalmic or carotid artery), damage to the photoreceptor layer prevails (Osborne et al., 1999, 2004). Although our observations need to be confirmed in a model of

chronic ocular hypertension, they provide a valid support for the involvement of the endocannabinoid system in processes that lead to RGC death in glaucoma.

The mechanisms responsible for the neuroprotective effects of (e)CBs in the retina are currently unknown. In the CNS, CB1 receptors are highly expressed on the presynaptic nerve endings of glutamatergic and GABAergic synapses (Twitchell et al., 1997; Davies et al., 2002), and this observation is consistent with numerous findings

from experimental studies, which suggest that CB1 receptor activation inhibits the presynaptic release of excitatory (glutamate) and inhibitory (GABA) neurotransmitters and — most importantly — that it prevents the massive release of glutamate under conditions of excitotoxicity (Kim and Thayer, 2000; Gilbert et al., 2007). Activation of CB1 receptors is believed to cause inhibition of voltagegated Ca2+ channels (Shen and Thayer, 1998; Gilbert et al., 2007) and activation of K+ channels (Gilbert et al., 2007). As a result, the influx of calcium ions following depolarization of the presynaptic neuron decreases, and this is accompanied by reduced exocytotic release of glutamate into the synaptic space.

Certain CBs that are already used in clinical practice for other purposes (e.g., THC) produce only a partial blockade of glutamatergic neurotransmission (Shen and Thayer, 1999; Gilbert et al., 2007), and this observation highlights one of the potential advantages of using CBs to combat excitotoxicity. These molecules might be used to selectively inhibit the glutamate release triggered by the pathologic stimulus, reducing the risk of serious adverse effects caused by the inhibition of physiologic release of this neurotransmitter. Molecules that produce complete inhibition of glutamatergic transmission are known to produce severe systemic effects. In addition to their effects on presynaptic glutamate release, CBs seem to also influence effects triggered by the activation of postsynaptic glutamate receptors. CB1 receptor

agonists reduce the influx of calcium ions triggered by stimulation of NMDA receptors in brain slices. This effect is abolished by SR14171A, pertussis toxin, and o-conotoxin, which blocks calcium channels of the PQ-type (Hampson et al., 1998). In our experimental model, we found that systemic administration of MK801, a use-dependent NMDA glutamate receptor antagonist, not only prevents ganglion-cell death (Nucci et al., 2005b) but reduces the increase in FAAH activity caused by acute ocular hypertension (Nucci et al., 2007b). These findings add further support to the hypothesis of a close correlation between excitotoxicity, retinal cell death, and the endocannabinoid system.

In spite of these findings, it would be an oversimplification to conclude that the neuroprotective effects of (e)CBs in the retina relate exclusively to their effects on the release and functions of glutamate. It is important to recall that the (e)CBs exert specific modulatory effects on vascular tone. Anandamide, for example, has recently been shown to induce dilation of the bovine ophthalmic artery (Romano and Lograno, 2006), and in healthy human subjects orally administered dronabinol (a synthetic delta-9-tetra- hydrocannabinol used to control chemotherapyrelated nausea and to stimulate appetite in cases of AIDS-induced anorexia) enhances retinal perfusion and markedly reduces IOP (Plange et al., 2007). The vasodilator effects of the eCBs seem to be related in part to their inhibition of endothelin-1