**4. Glutamate**

Neuroprotection is the strategy to prevent retinal ganglion cell death. There have been several methods, many still experimental, aimed at reducing glutamate excitotoxicity, nitric oxide, free radical production and tumour necrosis factor (TNF) inhibition [1-4,6]. With the latest research, glaucoma, which was once thought to be an optic neuropathy, then a retinal disease, is now being considered a neurodegenerative disease, like alzheimer and parkinson [7].

This chapter will review the present pathophysiology theories of neurodegeneration in glaucoma and highlight the latest updates in neuroprotection strategies, mechanisms that block apoptosis and improving the survival and functionality of the retinal ganglion cell.

The neurodegeneration seen in glaucoma is as an end result of apoptosis (programmed cell death) of the retinal ganglion cell (RGC). When the retinal ganglion cell dies, there is a degenerative change along the axon with the resulting clinical findings including thinning of the retinal nerve fiber layer (objectively measured by Optical Coherence Tomography, Heidelberg Retinal Tomography or GDx) and increased optic disc cupping. Retinal ganglion cell apoptosis results in visual field loss and ultimately loss of vision in glaucoma. There are several etiologies for retinal ganglion cell (RGC) death which occurs with and without elevated

The glutamate and nitric oxide (NO) theories were the early proposed mechanisms for neurodegeneration. There is a proposed oxidative component which results in oxidative stress

Although neurodegeneration theories were considered because of progression despite normal IOPs, increased IOP does have a role in RGC death. Increased IOP can block axonal transport of the excitotoxic transmitter, glutamate, at the level of the lamina cribrosa, leading to depri‐

Retinal ganglion cell apoptosis is thought to be a result of several factors:

**•** oxidative stress: free radical induced apoptosis (nitric oxide)

on the RGC due to increased IOP and hypoxia leading to apoptosis.

**2. Retinal ganglion cell death**

204 Glaucoma - Basic and Clinical Aspects

**•** increased intraocular pressure (IOP)

**•** neurotrophic factors deprivation

**3. Increased intraocular pressure**

**•** abnormal immune response

intraocular pressures.

**•** glutamate excitotoxicity

**•** glial cell activation

**•** hypo perfusion

Glutamate is an excitatory neurotransmitter that is continuously released by photoreceptors and OFF bipolar cells in the dark which results in the dark current. Light stimuli starts the process of phototransduction which leads to reduced glutamate concentration in the synaptic cleft. Glutamate transporters allow for the uptake of glutamate by müller cells which is converted by glutamine synthetase into glutamine which is then released by the glial cells. This glutamine is taken up by the neurons and hydrolysed by glutaminase to glutamate again. Glutamate allows the influx of calcium, resulting in high intracellular calcium levels which promote apoptosis. Glutamate in excess is neurotoxic, due to its induced excitotoxicity. The glutamate-glutamine cycle allows for natural homeostasis between the neurons and the glial cells (Figure 1).

Glutamate is released from degenerating cells or reduced uptake from müller's cells can increase the presence of glutamate. RGC may undergo apoptosis directly because of increased glutamate excitotoxicity. Müller cells can be injured by the excess glutamate which results in a secondary RGC death [16].

Glutamate ionotropic receptors are found on the post synaptic bipolar, horizontal, amacrine and ganglion cells. They are gating cation channels that are classified into 3 groups; N-methyl-D-aspartate (NMDA) receptors, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and kainite receptors. In view of this glutamate receptor antagonists have been found to reduce the neurotoxic effect of increased glutamate levels.

**5.1. Memantine**

**5.2. Eliprodil**

**5.3. Nitric oxide**

*5.3.1. Oxidative stress*

Memantine, is a NMDA receptor blocker which is approved in the USA for dementia associ‐ ated with alzheimer's disease. Although oral memantine clinically showed a protective effect on visual function and structural damage on macaque monkeys, it did not persist in long term treatment (>5 months) on ERG findings [25,26]. It has been used at doses of 4mg/kg po daily reaching concentrations of 0.3-1.8uM in the monkey vitreous [25,26]. The second phase III clinical trial showed that although the progression of disease was significantly lower in patients receiving the higher dose of memantine compared to patients receiving the low dose of memantine, there was no significant benefit compared to patients receiving placebo [27]. Latest animal studies have shown that using memantine in monkeys will result in an overall higher mean multifocal visual evoked potential (VEP) amplitudes than the non treated memantine monkeys when experimental glaucoma has been induced [28]. However it was not significant from baseline in the former. The use of the GDx in future studies will also allow more sensitive changes in retinal nerve fiber layer to be detected however, this may not directly be translated into functional damage, which in humans can be assesed with visual fields.

Strategies for Neuroprotection in Glaucoma http://dx.doi.org/10.5772/53776 207

This NMDA antagonist acts at the polyamine binding site of the NMDA receptor (NR2B subunit), blocking voltage dependent calcium channels. It has been shown to be neuroprotec‐ tive in cultured neurons of brain and retina from excitotoxic and ischemia damage at doses of 1-10mg/kg [29]. Eliprodil has shown reduction in the NMDA currents by 78% in a glutamate induced cytotoxicity model [30]. Although there has been promise of this drug in animal

Nitric oxide (NO) is a neurotransmitter, vasodilator and neuromodulator and can be neurotox‐ ic. Nitric oxide is found at the post junctional area of glutaminergic junctions (rods, bipolar, amacrine and ganglion cells) and acts as an intracellular mediator for glutamate. Excessive productionofnitricoxidebyastrocyteshasbeenshowntoplayaroleincelldeathinboththeoptic nerve head and the RGC [2,3,6,31,32]. Reactive oxidative species (ROS) may play a role in

Nitric oxide is produced by nitric oxide synthase (NOS-2). NOS has 3 isoforms inducible NO (iNO), endothelial NO (eNO) and neuronal NO (nNO). These oxidize L-arginine to L-citrulline, producing NO. Nitric oxide freely diffuses to adjacent neurons and combines with O2 – to form peroxynitrite anions (ONOO-) which is a potential toxin, setting into motion neuronal apoptosis. It can be induced by injury or cytokines, such as interleukin 1 beta, tumour necrosis factor alpha, resulting in high concentrations of nitric oxide [32,33]. Increased levels of NOS are seen in the optic nerve head of glaucoma patients [32]. Tumour necrosis factor (TNF) α is upregulated in the glaucomatous optic nerve head and induces NOS in the astrocytes [34].

studies, clinical trials have not been undertaken for glaucoma in humans.

neurodegeneration as a result of apoptosis (Figure 2).

Figure 1. =: The glutamate-glutamine cycle (RGC= Retinal Ganglion cell, GLAST = Glutamate Aspartate transporter, EAAT-1 =Excitatory Amino Acid Transporter)) **Figure 1.** The glutamate-glutamine cycle (RGC= Retinal Ganglion cell, GLAST = Glutamate Aspartate transporter, EAAT-1 =Excitatory Amino Acid Transporter))

Glutamate is released from degenerating cells or reduced uptake from müller's cells can increase the presence of glutamate. RGC may undergo apoptosis directly because of increased glutamate excitotoxicity. Müller cells can be injured by the excess glutamate which results in a secondary RGC death [16]. Glutamate ionotropic receptors are found on the post synaptic bipolar, horizontal, amacrine and ganglion cells. They are gating cation channels that are classified into 3 groups; N-methyl-D-aspartate (NMDA) receptors, α-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA) receptors and kainite receptors. In view of this glutamate receptor antagonists have been found Increased glutamate has been noted in the vitreous of glaucoma patients [17]. However the glutamate transporters; glutamate aspartate transporter (GLAST) and excitatory amino acid transporter-1 (EAAT-1) are localized exclusively to müller's cells and glutamate transporter -1 (GLT-1) and excitatory amino acid transporter- 2 (EAAT-2) in the brain, decreased with increasing IOPs [18,19,20] (Figure 1). Therefore, the increase IOP effect on the glutamate transporters can further aggravate glutamine neurotoxicity.

to reduce the neurotoxic effect of increased glutamate levels. Increased glutamate has been noted in the vitreous of glaucoma patients [17]. However the glutamate transporters; glutamate aspartate transporter (GLAST) and excitatory amino acid transporter-1 (EAAT-1) are localized exclusively to müller's cells and glutamate transporter -1 (GLT-1) and excitatory amino acid transporter- 2 (EAAT-2) in the brain, decreased with increase in Mice deficient in excitatory amino acid carrier-1 (EAAC1) or GLAST had RGC apoptosis in the absence of elevated IOP. Neuronal EAAC1 does not play a direct role in glutamate transport but transports cysteine much more than GLAST. This is important for the glutathione synthe‐ sis. Lack of glutathione made the RGCs more susceptible to oxidative stress [21].

elevated IOPs [18,19,20] (Figure 1). Therefore, the increase IOP effect on the glutamate transporters can further aggravate glutamine

eg alzheimer's and parkinson's disease as seen in rat and human models [22,23,24] . Glutamate opens calcium and sodium channels after binding to the NMDA receptor, which results in a high intracellular influx of calcium which starts the cascade of

Memantine, is a NMDA receptor blocker which is approved in the USA for dementia associated with alzheimer's disease. Although oral memantine clinically showed a protective effect on visual function and structural damage on macaque monkeys, it did not persist in long term treatment (>5 months) on ERG findings [25,26]. It has been used at doses of 4mg/kg po daily reaching concentrations of 0.3-1.8uM in the monkey vitreous [25,26]. The second phase III clinical trial showed that although the progression of disease was significantly lower in patients receiving the higher dose of memantine compared to patients receiving the low dose

Latest animal studies have shown that using memantine in monkeys will result in an overall higher mean multifocal visual evoked potential (VEP) amplitudes than the non treated memantine monkeys when experimental glaucoma has been induced [28] . However it was not significant from baseline in the former. The use of the GDx in future studies will also allow more sensitive changes in retinal nerve fiber layer to be detected however, this may not directly be translated into functional damage, which in

apoptosis. Therefore, NMDA receptor blockers have been investigated for counteracting possible glutamate excitotoxicity.

#### neurotoxicity. **5. Glutamate receptor antagonists**

**5.1. Memantine** 

humans can be assesed with visual fields.

Mice deficient in excitatory amino acid carrier-1 (EAAC1) or GLAST had RGC apoptosis in the absence of elevated IOP. Neuronal EAAC1 does not play a direct role in glutamate transport but transports cysteine much more than GLAST. This is important for the glutathione synthesis. Lack of glutathione made the RGCs more susceptible to oxidative stress [21]. **5. Glutamate receptor antagonists**  The NMDA ionotropic glutamate receptor has been shown to have an important role in the mechanisms of certain CNS disorders, The NMDA ionotropic glutamate receptor has been shown to have an important role in the mechanisms of certain CNS disorders, eg alzheimer's and parkinson's disease as seen in rat and human models [22,23,24]. Glutamate opens calcium and sodium channels after binding to the NMDA receptor, which results in a high intracellular influx of calcium which starts the cascade of apoptosis. Therefore, NMDA receptor blockers have been investigated for coun‐ teracting possible glutamate excitotoxicity.

of memantine, there was no significant benefit compared to patients receiving placebo [27].

### **5.1. Memantine**

(AMPA) receptors and kainite receptors. In view of this glutamate receptor antagonists have

Hydrolysed by glutaminase

**Figure 1.** The glutamate-glutamine cycle (RGC= Retinal Ganglion cell, GLAST = Glutamate Aspartate transporter,

Increased glutamate has been noted in the vitreous of glaucoma patients [17]. However the glutamate transporters; glutamate aspartate transporter (GLAST) and excitatory amino acid transporter-1 (EAAT-1) are localized exclusively to müller's cells and glutamate transporter -1 (GLT-1) and excitatory amino acid transporter- 2 (EAAT-2) in the brain, decreased with increasing IOPs [18,19,20] (Figure 1). Therefore, the increase IOP effect on the glutamate

Mice deficient in excitatory amino acid carrier-1 (EAAC1) or GLAST had RGC apoptosis in the absence of elevated IOP. Neuronal EAAC1 does not play a direct role in glutamate transport but transports cysteine much more than GLAST. This is important for the glutathione synthe‐

sis. Lack of glutathione made the RGCs more susceptible to oxidative stress [21].

may undergo apoptosis directly because of increased glutamate excitotoxicity. Müller cells can be injured by the excess glutamate

Glutamine (RGC)

Glutamine (Muller's cells)

eg alzheimer's and parkinson's disease as seen in rat and human models [22,23,24] . Glutamate opens calcium and sodium channels after binding to the NMDA receptor, which results in a high intracellular influx of calcium which starts the cascade of

Memantine, is a NMDA receptor blocker which is approved in the USA for dementia associated with alzheimer's disease. Although oral memantine clinically showed a protective effect on visual function and structural damage on macaque monkeys, it did not persist in long term treatment (>5 months) on ERG findings [25,26]. It has been used at doses of 4mg/kg po daily reaching concentrations of 0.3-1.8uM in the monkey vitreous [25,26]. The second phase III clinical trial showed that although the progression of disease was significantly lower in patients receiving the higher dose of memantine compared to patients receiving the low dose

Latest animal studies have shown that using memantine in monkeys will result in an overall higher mean multifocal visual evoked potential (VEP) amplitudes than the non treated memantine monkeys when experimental glaucoma has been induced [28] . However it was not significant from baseline in the former. The use of the GDx in future studies will also allow more sensitive changes in retinal nerve fiber layer to be detected however, this may not directly be translated into functional damage, which in

apoptosis. Therefore, NMDA receptor blockers have been investigated for counteracting possible glutamate excitotoxicity.

the glutathione synthesis. Lack of glutathione made the RGCs more susceptible to oxidative stress [21].

The NMDA ionotropic glutamate receptor has been shown to have an important role in the mechanisms of certain CNS disorders, eg alzheimer's and parkinson's disease as seen in rat and human models [22,23,24]. Glutamate opens calcium and sodium channels after binding to the NMDA receptor, which results in a high intracellular influx of calcium which starts the cascade of apoptosis. Therefore, NMDA receptor blockers have been investigated for coun‐

of memantine, there was no significant benefit compared to patients receiving placebo [27].

been found to reduce the neurotoxic effect of increased glutamate levels.

Acid Transporter))

EAAT-1 =Excitatory Amino Acid Transporter))

Glutamate uptake by glutamate transporters (GLAST) & EAAT--1

206 Glaucoma - Basic and Clinical Aspects

neurotoxicity.

**5.1. Memantine** 

which results in a secondary RGC death [16].

Glutamate (Muller's cell)

Glutamate (RGC)

**5. Glutamate receptor antagonists** 

**5. Glutamate receptor antagonists**

teracting possible glutamate excitotoxicity.

humans can be assesed with visual fields.

to reduce the neurotoxic effect of increased glutamate levels.

transporters can further aggravate glutamine neurotoxicity.

Memantine, is a NMDA receptor blocker which is approved in the USA for dementia associ‐ ated with alzheimer's disease. Although oral memantine clinically showed a protective effect on visual function and structural damage on macaque monkeys, it did not persist in long term treatment (>5 months) on ERG findings [25,26]. It has been used at doses of 4mg/kg po daily reaching concentrations of 0.3-1.8uM in the monkey vitreous [25,26]. The second phase III clinical trial showed that although the progression of disease was significantly lower in patients receiving the higher dose of memantine compared to patients receiving the low dose of memantine, there was no significant benefit compared to patients receiving placebo [27].

Latest animal studies have shown that using memantine in monkeys will result in an overall higher mean multifocal visual evoked potential (VEP) amplitudes than the non treated memantine monkeys when experimental glaucoma has been induced [28]. However it was not significant from baseline in the former. The use of the GDx in future studies will also allow more sensitive changes in retinal nerve fiber layer to be detected however, this may not directly be translated into functional damage, which in humans can be assesed with visual fields.

#### **5.2. Eliprodil**

Figure 1. =: The glutamate-glutamine cycle (RGC= Retinal Ganglion cell, GLAST = Glutamate Aspartate transporter, EAAT-1 =Excitatory Amino Glutamate is released from degenerating cells or reduced uptake from müller's cells can increase the presence of glutamate. RGC This NMDA antagonist acts at the polyamine binding site of the NMDA receptor (NR2B subunit), blocking voltage dependent calcium channels. It has been shown to be neuroprotec‐ tive in cultured neurons of brain and retina from excitotoxic and ischemia damage at doses of 1-10mg/kg [29]. Eliprodil has shown reduction in the NMDA currents by 78% in a glutamate induced cytotoxicity model [30]. Although there has been promise of this drug in animal studies, clinical trials have not been undertaken for glaucoma in humans.

#### **5.3. Nitric oxide**

Glutamate ionotropic receptors are found on the post synaptic bipolar, horizontal, amacrine and ganglion cells. They are gating cation channels that are classified into 3 groups; N-methyl-D-aspartate (NMDA) receptors, α-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA) receptors and kainite receptors. In view of this glutamate receptor antagonists have been found Increased glutamate has been noted in the vitreous of glaucoma patients [17]. However the glutamate transporters; glutamate aspartate transporter (GLAST) and excitatory amino acid transporter-1 (EAAT-1) are localized exclusively to müller's cells and Nitric oxide (NO) is a neurotransmitter, vasodilator and neuromodulator and can be neurotox‐ ic. Nitric oxide is found at the post junctional area of glutaminergic junctions (rods, bipolar, amacrine and ganglion cells) and acts as an intracellular mediator for glutamate. Excessive productionofnitricoxidebyastrocyteshasbeenshowntoplayaroleincelldeathinboththeoptic nerve head and the RGC [2,3,6,31,32]. Reactive oxidative species (ROS) may play a role in neurodegeneration as a result of apoptosis (Figure 2).

#### glutamate transporter -1 (GLT-1) and excitatory amino acid transporter- 2 (EAAT-2) in the brain, decreased with increase in elevated IOPs [18,19,20] (Figure 1). Therefore, the increase IOP effect on the glutamate transporters can further aggravate glutamine *5.3.1. Oxidative stress*

Mice deficient in excitatory amino acid carrier-1 (EAAC1) or GLAST had RGC apoptosis in the absence of elevated IOP. Neuronal EAAC1 does not play a direct role in glutamate transport but transports cysteine much more than GLAST. This is important for The NMDA ionotropic glutamate receptor has been shown to have an important role in the mechanisms of certain CNS disorders, Nitric oxide is produced by nitric oxide synthase (NOS-2). NOS has 3 isoforms inducible NO (iNO), endothelial NO (eNO) and neuronal NO (nNO). These oxidize L-arginine to L-citrulline, producing NO. Nitric oxide freely diffuses to adjacent neurons and combines with O2 – to form peroxynitrite anions (ONOO-) which is a potential toxin, setting into motion neuronal apoptosis. It can be induced by injury or cytokines, such as interleukin 1 beta, tumour necrosis factor alpha, resulting in high concentrations of nitric oxide [32,33]. Increased levels of NOS are seen in the optic nerve head of glaucoma patients [32]. Tumour necrosis factor (TNF) α is upregulated in the glaucomatous optic nerve head and induces NOS in the astrocytes [34].

*5.3.3. The future*

tective effects [42,43,44].

**6.1. Brimonidine**

**7.1. Betaxolol**

with the a carbonic anhydrase inhibitor alone [40].

**6. Alpha adrenergic receptor agonist**

methyl-D-aspartate receptors [43,46-48].

**7. Selective beta receptor blockade**

Krauss 2011 and Impagnatiello 2011 have had success in lowering IOP in preclinical trials with a nitric oxide donating prostaglandin F2 agonist (BOL-303259-X) more than with latanoprost (prostaglandin F2 agonist) alone [38,39]. Fabrizi 2012, also had some success with combining a carbonic anhydrase inhibitor with a nitric oxide moiety, NCX250 in lowering IOP compared

Strategies for Neuroprotection in Glaucoma http://dx.doi.org/10.5772/53776 209

α2 adrenergic agonists are a known group of anti glaucoma drugs that inhibit adenylate cyclase, reducing cAMP, thereby decreasing aqueous production. They also act by increasing uveoscleral outflow. α2A receptors can be found in non pigmented ciliary epithelium, α2B receptors on neuronal dendrites and α2C receptors on photoreceptors cell bodies and inner segments [41]. α<sup>2</sup> agonists have been shown to have secondary neuropro‐

α2 adrenergic receptors can modulate the release of neurotransmitters such as glutamate [45]. NMDA receptors when stimulated results in an increase in intracellular Ca2+ and an inward current in the RGC. Brimonidine, an α2 agonist, can block the NMDA receptors which results in controlling the intracellular calcium, hereby allowing neuroprotection [23,46]. Brimonidine is also thought to up regulate brain derived neurotrophic factor (BDNF), activating anti apoptotic genes and the cell survival signaling pathway. It is also thought to modulate the N

Brimonidine is also known to upregulate not only BDNF, but prosurvival factors, such as anti apoptotic factors B-cell lymphoma -2 (Bcl-2) and B-cell lymphoma extra large (bcl-xl), basic fibroblastic growth factor (bFGF) and extracellular signal regulated kinases (ERKs). These

Beta blockers have a long history of use in reducing the IOP in glaucoma by reducing the production of aqueous humour. Levobetaxolol, timolol and metipranolol have been shown to have secondary neuroprotective effect by reducing sodium and calcium influx, which reduces the release of glutamate with levobetaxolol being more effective than timolol [50,51,52].

Betaxolol has been shown to reduce the spontaneous firing rate by suppressing glutamategated current and in effect Na currents in the ganglion cells [52]. By doing this it also reversibly

actions assist in the prevention of neuronal death and promotes cell survival [49].

**Figure 2.** Multiple mechanisms for neurodegeneration which may be aggravated by vascular dysregulation, hypoxia and elevated IOP

nNO and iNO are expressed in reactive astrocytes. Increased NO reacts with a superoxide anion which can be toxic to the axons of the retinal ganglion cell. Motallebipour et al, showed a genetic association between iNO and primary open angle glaucoma (POAG) using genetic analysis and nuclear factor [35]. iNO is located in the astrocytes and microglial in the optic nerve head and expresses more activity with exposure to increased intraocular pressure and cytokines. This results in increased in NO production and the induction of the apoptotic cascade [36]. The NO oxide has its effect in both the astrocytes of the optic nerve head and the pericytes of the vasculature [32].

#### *5.3.2. Vascular modulation*

The endothelial NO synthase (eNOS) is expressed in the trabecular membrane and schlem's canal cells. eNOS produces nitric oxide which regulates the vascular tone causing smooth muscle relaxation and relaxation of the trabecular meshwork which improves aqueous humour outflow [37]. Elevation of the IOP increases the shear stress which activates eNOS which results in increase in the pressure dependent outflow.

### *5.3.3. The future*

Krauss 2011 and Impagnatiello 2011 have had success in lowering IOP in preclinical trials with a nitric oxide donating prostaglandin F2 agonist (BOL-303259-X) more than with latanoprost (prostaglandin F2 agonist) alone [38,39]. Fabrizi 2012, also had some success with combining a carbonic anhydrase inhibitor with a nitric oxide moiety, NCX250 in lowering IOP compared with the a carbonic anhydrase inhibitor alone [40].
