Possible role of excitotoxicity in the pathogenesis of glaucoma

Clinical and Experimental Ophthalmology

2006;

34

: 54–63doi:10.1111/j.1442-9071.2006.1146.x

© 2006 Royal Australian and New Zealand College of Ophthalmologists

�

Correspondence:

Assistant Professor Robert J Casson, Department of Ophthalmology and Visual Science, Royal Adelaide Hospital, North Tce, Adelaide, SA

5000, Australia. Email: [email protected]

Received 5 June 2005; accepted 10 October 2005.

Perspective

Possible role of excitotoxicity in the pathogenesis of glaucoma

Robert J Casson

DPhil FRANZCO

Department of Ophthalmology and Visual Science, Royal Adelaide Hospital, Adelaide, South Australia, Australia

that excessive extracellular glutamate levels can be toxic toneurones, including those in the retina, and that excitotox-icity may play a role in the pathogenesis of certain neuro-logical diseases.

5

Overview of neurotransmission

Neurones, including those in the retina and visual pathways,are anatomically and functionally specialized for the electri-cal and chemical transmission of information. These trans-missions allow complex communication and data processing,resulting in the visual perception of the external environ-ment. The chemicals that transmit information between neu-rones are termed neurotransmitters. They are released fromneurones, act on membrane-bound receptors and producefunctional changes in the target cell. The general mecha-nisms of chemical synaptic transmission are shown in Fig. 1.

Axons at rest are electrically polarized, exhibiting a neg-ative resting membrane potential of approximately

−

60 mV(inside

vs

. outside the cell). In the generation of an actionpotential, positively charged ions (cations) enter the cell,and the polarization is removed (depolarization). A train ofaction potentials is produced as the cell depolarizes, lead-ing to propagation of the nerve impulse and release ofneurotransmitter.

The effect that a given neurotransmitter has on the post-synaptic neurone depends on the sequence of changes thatit induces in the cell. Neurotransmitters are generally eitherexcitatory or inhibitory, the former depolarizing the cell andinducing a train of action potentials, the latter hyperpolariz-ing the cell. The principle excitatory neurotransmitter in theretina is the amino acid glutamate, which acts as a ligand ata number or receptor subtypes.

Excitatory amino acid receptors

Excitatory amino acid receptors are currently divided intothree large families:

N

-methyl-

D

-aspartate (NMDA) recep-tors,

α

-amino-3-hydroxy-5-methyl-4-isoxazole propionate(AMPA)/kainate receptors and metabotropic receptors.

A

BSTRACT

Excitotoxicity describes the process of neuronal injury byexcess stimulation of amino acid receptors. This form of insultwas first described in the retina, and subsequently has beenshown to be an important component of the pathogenesisof ischaemic and traumatic injury in the central nervous sys-tem. Furthermore, there is increasing evidence that excito-toxicity is involved in several chronic neurological conditions,and anti-excitotoxic treatment has already been approved forsome of these conditions. A large-scale trial is currently under-way that will determine the efficacy of an anti-excitotoxic drug(memantine) in the management of glaucoma. This reviewprovides an overview of neurotransmission and the mecha-nisms of excitotoxicity. The evidence for excitotoxicity as acomponent of certain neurological diseases, including glau-coma, is discussed.

Key words:

excitotoxicity

,

glaucoma

,

memantine

,

opticnerve

.

B

ACKGROUND

Definition of excitotoxicity

In 1957, Lucas and Newhouse hypothesized that the neona-tal mouse retina might be affected by a variety of agents of‘biochemical importance’: a striking finding was a loss ofneurones in the inner layers of the retina after subcutaneousglutamate administration.

1,2

Although the mechanism wasnot understood, this was the first demonstration of an exci-totoxic insult. In 1959, Curtis

et al.

described the depolariz-ing effect of glutamate on rat spinal neurones,

3

and thenotion of an excitatory amino acid neurotransmitter wasdeveloped. A decade later, Olney described brain lesions inmice treated with monosodium glutamate, and he coined theterm ‘excitoxicity’ to describe the process of neuronal deathcaused by excessive or prolonged activation of receptors forexcitatory amino acid neurotransmitters.

4

Subsequently, alarge body of evidence has supported this finding, indicating

Excitotoxicity in glaucoma pathogenesis 55


NMDA and AMPA/kainate receptors are ligand-gated ionchannels and mediate fast synaptic transmission byglutamate. NMDA receptors permit the influx of sodiumions (Na

+

) and calcium ions (Ca

2

+

) and the efflux of potas-sium ions (K

+

). At resting membrane potentials, NMDAreceptors are blocked by magnesium ions (Mg

2

+

), preventingtheir activation by glutamate (Fig. 2). Therefore, they areprobably not involved in primary synaptic transmission atglutamatergic synapses. However, the blockade by Mg

2

+

ions is voltage-dependent; hence, they can be activated byglutamate after the neurone has been depolarized byanother excitatory mechanism.

6

Five subunits of the NMDA

receptor have been described: the obligatory NR1 subunittogether with the modulatory NR2A–NR2D subunits.

7,8

Subunits of the NMDA receptor have been immunohis-tochemically localized to the inner retina of rodents, par-ticularly to retinal ganglion cell (RGC) and amacrine cellbodies and the inner plexiform layer.

9

Immunohistochemicalstudy of human eyes indicates that there are distinct expres-sion profiles of the different subunits on RGC bodies, withNR1, NR2C and NR2D predominating.

10

NMDA receptorsubunits have been detected on cortical astrocytes,

11,12

andmay, by extrapolation, be present on optic nerve head astro-cytes. The NR1 subunit has also been located on the intraor-bital rat optic nerve.

13

However, to my knowledge, thepresence of NMDA receptors on unmyelinated optic nerveaxons in the retina and/or optic nerve head (optic nervehead refers to the portion of the optic nerve as it exits theglobe, in particular, the prelaminar and laminar portions) hasnot been reported.

AMPA/kainate receptors can be activated by glutamate,AMPA and kainic acid. They permit the influx of Na

+

ionsand the efflux of K

+

ions. They are thought to mediate rapidexcitatory transmission at most glutamatergic synapses.There is evidence that oligodendrocytes (myelin-formingcells in the central nervous system [CNS]) have AMPA/kainate receptors and that overstimulation of these receptorscan result in white matter injury.

14,15

In addition, there isevidence that spinal cord astrocytes have AMPA/kainatereceptors.

16

In one study, inhibition of AMPA/kainate recep-tors after optic nerve crush injury attenuated the injury morethan inhibition of NMDA receptors;

15

however, the AMPA/kainate receptors have not been identified on unmyelinatedRGC axons.

Metabotropic receptors are G-protein-linked receptorsand are not considered to be involved in fast synaptic trans-mission; however, they may play a minor role in grey matterexcitotoxic injury, and perhaps a more significant role inwhite matter injury.

17,18

Glutamate uptake

In normal synaptic functioning, the excitatory action ofglutamate is rapidly terminated due to its efficient removalfrom the synapse by uptake mechanisms in glia and nerveterminals.

19

Specific transporter proteins co-transportglutamate with Na

+

while counter-transporting K

+

, using thetransmembrane electrochemical gradients for the Na

+

and K

+

ions as a driving force. Although astrocytes are important forglutamate uptake in the CNS, Müller cells predominantlysubserve this function in the retina. After its uptake by glialcells, glutamate is converted to glutamine, released into theextracellular space and taken up by neurones, where it isreconverted to glutamate (Fig. 1). Hence, this neuronal-glialmetabolic shunt forms an important component of excitatoryneurotransmitter metabolism. Müller cells protect RGCsagainst excitotoxic injury in culture.

20

Furthermore, dysfunction of this system could conceiv-ably lead to excitotoxic injury.

21

Figure 1.

Overview of glutamate (Glu) neurotransmission.Glutamate is synthesized in the presynaptic neurone and stored interminal vesicles. Upon depolarization, there is Ca

2

+

-dependentrelease of glutamate into the synaptic cleft. Glutamate binds topost-synaptic receptors, causing site-dependent effects, but usuallycausing influx of Na

+

and Ca

2

+

and depolarization. Extracellularglutamate is taken up by glial cells and converted to glutamine(Gln), then re-taken by neurones.

Presynaptic neurone

Glu

Glu

GluGln

Gln

Glial cell

Post-synaptic neurone

Post-synaptic receptor

Vesicular transporter

Figure 2.

Schematic of the

N

-methyl-

D

-aspartate (NMDA)receptor. NMDA receptors permit the influx of Na

+

and Ca

2

+

ionsand the efflux of K

+

ions. At resting membrane potentials, NMDAreceptors are blocked by Mg

2

+

ions, preventing their activation byglutamate. The blockade by Mg

2

+

ions is voltage-dependent; hence,they can be activated by glutamate after the neurone has beendepolarized by another excitatory mechanism. Five subunits of theNMDA receptor have been described: the obligatory NR1 subunittogether with the modulatory NR2A–NR2D subunits. An extracel-lular glycine binding site is present and memantine can block thechannel in an uncompetitive manner.

Na+

Ca2+

Mg2+

Memantine binding site

Glutamate/NMDA binding siteGlycine binding site

Schematic of the NMDA receptor

56 Casson


M

ECHANISMS

OF

EXCITOTOXICITY

Classical excitotoxicity

Excitotoxic injury involves a self-reinforcing cascade ofevents that involves loss of ionic homeostasis and ultimatelycell death. The principle ionic changes that are involved inthe molecular pathology are: (i) an initial influx of Na

+

ions;and (ii) an increase in intracellular Ca

2

+

ion. These changesoccur in parallel and result in ongoing depolarization, ampli-fication and continuation of the injury by further release ofglutamate, oncotic/necrotic death and calcium-induceddeath processes. These processes are shown in Fig. 3.

Depolarization is initiated primarily by activation ofAMPA receptors and subsequently by activation of voltage-dependent Na

+

channels. Chronic depolarization causes lossof the Mg

2

+

blockade of the NMDA receptor and influx ofCa

2

+

through this channel. The initial Na

+

influx is followedby passive influx of chloride ions (Cl

–

) along their electro-chemical gradient and influx of water along the osmoticgradient. This increase in intracellular water can lead toswelling of intracellular organelles and eventually cell lysisin a necrotic-style death, with release of intracellular con-

tents, including more glutamate into the extracellularmilieu.

Loss of cellular calcium homeostasis plays a critical rolein cell death. During excitotoxic injury, a number of inter-dependent, self-reinforcing processes lead to a rise in intra-cellular Ca

2

+

. These processes include: (i) Ca

2

+

influx throughvoltage-sensitive calcium channels due to membrane depo-larization; (ii) glutamate release causing Ca

2

+

influx throughNMDA channels (and through kainate/AMPA receptorchannels);

22

(iii) reversal of the membrane Ca

2

+

/Na

+

trans-porter that is normally driven by the transmembrane Na

+

concentration gradient;

23

(iv) failure of Ca

2

+

extrusionbecause of adenosine triphosphate (ATP) depletion;

24

(v) therelease of calcium from intracellular stores and by inositoltriphosphate formed by metabotropic glutamate receptorstimulation;

25

and (vi) the increase in intracellular Ca

2

+

lead-ing to mitochondrial dysfunction, further exacerbating ATPdepletion.

26,27

The elevation of intracellular calcium levels is thought tobe a major component of cell death.

28

The increase in Ca

2

+

has a number of deleterious consequences, including theactivation of destructive lipases, proteases and nucleases;production of free radicals, mitochondrial injury; and theCa

2

+

-dependent exocytosis of vesicular glutamate from thenerve terminals.

28

Furthermore, Ca

2

+

-induced cellular demiseis triggered more efficiently when influx occurs through cer-tain channels rather than others: influx through the NMDAreceptor is particularly efficient at causing cell death. Thisphenomenon has been termed the ‘source-specificity’hypothesis of Ca

2

+

neurotoxicity.

29

Slow excitotoxicity and metabolic impairment

Energy failure secondary to mitochondrial dysfunction ren-ders neurones vulnerable to excitotoxic injury.

30,31

DecliningATP levels lead to failure of the ionic pumps, in particular,the Na

+

/K

+

pump that is responsible for maintaining theresting membrane potential. A slowly increasing membranepotential increases the likelihood of depolarization, releaseof the voltage-dependent Mg

2

+

block on the NMDAchannel

30,31

and entry into the self-reinforcing excitotoxicitycascade as described. In slow excitotoxicity, ambient concen-trations of glutamate are sufficient to produce excitotoxicity.

Free radicals

Normally the rates of free radical production and eliminationwithin a cell are balanced: molecular oxygen (O

2

) is con-verted to superoxide (O

2–

) by oxidative enzymes in theendoplasmic reticulum, mitochondria, plasma membrane,peroxisomes and cytosol. O

2–

is converted to H

2

O

2

by dis-mutation and thence to OH• by the Cu

2

+

/Fe

2

+

-catalsyedFenton reaction. H

2

O

2

is also derived from oxidases in per-oxisomes. The major anti-oxidant enzymes in the retina aresuperoxide dismutase, catalase and glutathione peroxidase.These enzyme systems comprise an important component ofendogenous neuroprotection in the retina. Excitotoxic-

Figure 3.

The excitotoxic cascade. During excitotoxic injury, anumber of interdependent, self-reinforcing processes lead to a risein intracellular Ca

2

+

. These processes include: (i) Ca

2

+

influxthrough voltage-sensitive calcium channels due to membranedepolarization; (ii) glutamate release causing Ca

2

+

influx through

N

-methyl-

D

-aspartate (NMDA) channels (and through kainate/

α

-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) recep-tor channels); (iii) reversal of the membrane Ca

2

+

/Na

+

transporterthat is normally driven by the transmembrane Na

+

concentrationgradient; (iv) failure of Ca

2

+

extrusion because of adenosine triph-osphate (ATP) depletion; and (v) the increase in intracellular Ca

2

+

leading to mitochondrial dysfunction, further exacerbating ATPdepletion, and to activation of nitric oxide synthase (NOS) anddestructive enzymes. The elevation of intracellular calcium levelsis thought to be a major component of cell death. The increase inCa

2

+

has a number of deleterious consequences, including the acti-vation of destructive lipases, proteases and nucleases; production offree radicals, mitochondrial injury; and the Ca

2

+

-dependent exocy-tosis of vesicular glutamate from the nerve terminals.

Mitochondrialmembrane dysfunction

DepolarizationK+ effluxNa+ infulx

NMDA recep .Mg2+blockremoved

NMDA recep .activation

Glutamate release

Voltage - gatedCa2+ channels open

Na+/Ca 2+

exchangeNa+/Ca2+

exchange

Ca2+

ATPasefailure

Ca2+Mitochondrial

membrane dysfunction

PhospholipasesPhospholipases

Proteases/caspases

Cell

death

NOS activation

The Excitotoxicity Cascade



induced Ca2+ overload causes a net increase in free radicals,overwhelming the anti-oxidant capabilities and contributingto the injury in the retina. Their principle injurious effectsare: (i) lipid peroxidation of membranes; (ii) oxidative mod-ification of proteins; and (iii) reactions with thymine innuclear and mitochondrial DNA, producing single-strandedbreaks.32 A principle means by which free radicals have beenimplicated in the pathogenesis of glaucoma is by their inter-action with nitric oxide (NO).33

Nitric oxide

Nitric oxide is an important neuromediator throughout theCNS, and is implicated in many physiological processes inthe retina.34 It is synthesized from L-arginine via the actionof nitric oxide synthase (NOS) and three distinct isoformsof NOS have been characterized. Neuronal NOS (nNOS)and endothelial NOS (eNOS) are Ca2+-dependent and areconstitutively expressed by a variety of nervous tissues andby endothelial cells of blood vessels, respectively.34 Induc-ible or immunological NOS (iNOS) is Ca2+-independentand is not generally found under normal physiologicalconditions.34

Although NO has important physiological functions inthe retina, it has been implicated in the pathogenesis ofexcitotoxicity-associated retinal injury (Fig. 4). Increases innNOS, generally in the inner retina, have been shown fol-lowing pressure-induced ischaemia35,36 and in the two-vesselocclusion model of ischaemia.37 Increases in eNOS havebeen detected after pressure-induced ischaemia38 and opticnerve ligation.39 Activated microglial cells as well as retinalastrocytes and Müller cells produce iNOS.40 Substantial ele-vations of iNOS in the retina have been shown using thehigh intraocular pressure (IOP) model of ischaemia,40 afteroptic nerve bundle occlusion,41 in a murine model ofischaemic proliferative retinopathy42 and in patients withdiabetic retinopathy and ocular ischaemic disease.43 Inhibi-tion of NOS,44 especially iNOS,40 leads to histological andfunctional protection of the ischaemic retina.

Furthermore, iNOS has been demonstrated in the opticnerve head region of glaucoma patients,33 and its inhibitionby aminoguanidine in experimental glaucoma has been sug-gested to be neuroprotective;33 however, this notion hasrecently been challenged.45

Primary versus secondary neuronal degeneration

Apart from rare neurological disorders such as neurolathy-rism,46 excitotoxic injury is generally a secondary phenome-non. The most common initial causes are ischaemia andtrauma, resulting in a release of glutamate from necrotic cellsand/or a failure of ionic homeostasis and initiation of thedepolarization-induced excitotoxicity cascade. Hence, notonly may neurones be killed by the initial ischaemic and/ortraumatic insult, but surviving neurones may subsequently beinjured by excessive extracellular glutamate levels and exci-totoxicity (in ischaemia, the excitotoxic cascade is also animportant component of the primary injury). This additionalinsult is often referred to as secondary degeneration, and ithas been a focus for neuroprotective strategies (the retro-grade loss of neuronal bodies after axonal injury is also some-times, confusingly, called secondary degeneration, but here,secondary degeneration refers only to the demise of neu-rones that were not initially involved).

THE ROLE OF EXCITOTOXICITY IN NEUROLOGICAL DISORDERS

Stroke

Excitotoxic injury is an intrinsic component of ischaemicbrain injury (Fig. 1). There is a massive increase in extracel-lular glutamate levels following experimental ischaemicinjury47 and many studies have shown that, at least in modelsof focal ischaemic injury, glutamate receptor inhibitors areneuroprotective against ischaemic injury.48 A large numberof clinical studies have attempted to translate success frombench to bedside, but the results have been disappointing.48

There are a number of possible explanations for this failure,including invalid animal models of stroke, timing and drugdelivery issues and adverse effects. At this time, the prospectfor a successful anti-excitotoxic agent to treat stroke appearsdismal.

Similarly, there is considerable experimental evidencethat inhibition of glutamate receptors can attenuateischaemic retinal injury,49 but no clinical studies have beenperformed.

Traumatic injury

Several studies have found that extracellular glutamate levelsare increased following experimental traumatic brain50 andspinal cord51–54 injury. Traumatic CNS injury causes excito-toxic injury in both the neuronal (grey matter) and axonal(white matter) tissues. There is evidence that macroglialcells, both oligodendrocytes and astrocytes, play an impor-

Figure 4. Mechanism of nitric oxide (NO) toxicity. Excitotoxicinjury causes an increase in intracellular Ca2+, which activates nitricoxide synthase (NOS). NO combines with superoxide to form thehighly reactive peroxynitrite (ONOO–) species. ONOO– causessingle-stranded breaks in DNA, which activate poly(adenosinediphosphate-ribose) polymerase (PARP), leading to nicotinamideadenine dinucleotide (NAD) and adenosine triphosphate (ATP)depletion.

NOS NO O2•-+ ONOO-

PARP

Excitotoxic injury

Single-stranded DNA breaks

Ca2+

NAD ATP

NOS NO O2•-+ ONOO-

PARP

Single-stranded DNA breaks

Ca2+

NAD ATP

58 Casson


tant role in white matter excitotoxic injury,55–57 and thatAMPA receptors rather than NMDA receptors areinvolved.55–57

Following spinal cord injury, glutamate is released fromneurones and damaged axons. The raised extracellularglutamate levels activate the excitotoxic cascade, furtherdamaging neuronal cell bodies. In addition, Na+ influx intoaxons occurs via dysfunctional Na+/K+ ATPase pumps andvoltage-gated Na+ channels.57 The elevated intracellular Na+

levels cause reversal of both the Na+/Ca2+ exchanger and theglutamate/Na+ exchanger channels; hence glutamate is fur-ther extruded from the axon and Ca2+ levels increase insidethe axon.58 The increased Ca2+ activates degradative pro-teins, including calpain, resulting in neurofilament degrada-tion and axonal dysfunction59,60 (Fig. 5). Concurrently, Ca2+

and Na+ influxes into myelin sheaths and oligodendrocytecell bodies via AMPA channels lead to oligodendrocytedeath.57

Several anti-excitotoxic drugs have been shown to atten-uate experimental CNS injury,61,62 but none have thus farbeen effective clinically.

Chronic neurological disease

Excitotoxicity has been implicated in the pathogenesis of anumber chronic neurodegenerative diseases, includingamyotrophic lateral sclerosis (ALS), Huntington’s disease,Parkinson’s disease and Alzheimer’s disease. In ALS, there isevidence for both NMDA- and AMPA/kainate-mediatedexcitotoxic injury.63 A glutamate release inhibitor and Na+

channel blocker, riluzole, has been shown in clinical studiesto improve both function and survival,64 and is used as atreatment for this disease. Slow excitotoxic injury in thepresence of energy failure may play a role in Huntington’sdisease,65 and anti-excitotoxic treatment is being evaluatedin clinical studies. There is evidence for excitotoxic injury inParkinson’ disease66 and several antiparkinsonian drugs, orig-inally introduced as supposed anticholinergic agents, such asprocyclidine, amantadine, budipine and memantine, are, infact, NMDA channel blockers.5 Hence, the beneficial effectof memantine on symptoms may relate to its anti-excitotoxic

effects. Similarly, memantine has been shown to attenuatesymptoms in moderate to severe Alzheimer’s disease, and iscurrently used to treat this disorder.67,68

EVIDENCE FOR EXCITOTOXICITY IN THE PATHOGENESIS OF GLAUCOMA

The pathogenesis of glaucoma remains unclear. Recent stud-ies have confirmed the importance of IOP in the progressionof the disease,69,70 but the exact causal relationship betweenIOP and the pressure-sensitive optic neuropathy remainsunclear. The two principle theories of glaucoma pathogene-sis remain the mechanical and vascular theories, each havingvied for supremacy for the past 150 years. However, thesetheories are not mutually exclusive; furthermore, an excito-toxic component is compatible with both of these theories,and could conceivably produce a secondary pathology thatis potentially treatable by pressure-independent strategies.

In the vascular theory, the optic nerve head is consideredto undergo a chronic ischaemic injury. There is considerableevidence for blood flow incompetence at the optic nervehead in glaucoma patients;71 and interestingly substantialevidence to indicate the presence of retinal blood flowabnormalities in glaucoma patients.72–78 This latter point isimportant because it implies that glaucoma may not only bea white matter disease, as it is usually considered, but mayalso primarily affect retinal cell bodies. Although RGCs aregenerally considered to be the only retinal cells that areaffected by glaucoma, implying that glaucoma is truly onlya white matter disease, a possible pathological effect onother inner retinal cell types, in particular amacrine cells,although suggested,79 has never been reported.

In the mechanical theory, there is a pressure-related injuryto the axons at the level of the optic nerve and/or an opticnerve head that is particularly susceptible to mechanicalstress. Hence, there is little room for primary retinal injuryin this theory, but RGC bodies would be expected to sufferretrograde injury. A related contemporary theory suggeststhat a reduction in target-derived trophic support plays apathogenic role.80,81 In this theory, a pressure-related reduc-tion in the retrograde supply of brain-derived trophic factorfrom the lateral geniculate body causes the RGCs to die.Here, the RGC bodies suffer from the lack of trophic supportand the axons suffer from the pressure-related injury andfrom the RGC body pathology.

Retinal ganglion cell loss is an intrinsic component ofglaucomatous optic neuropathy: clinically, RGC axons arelost at the level of the optic nerve head and retinal nervefibre layer; pathologically, loss of the RGC bodies in theganglion cell layer is a characteristic finding. If glaucoma isa ‘pure’ white matter disease, then this loss of the RGC bodiesmust simply be occurring due to retrograde degeneration.When the intraorbital optic nerve is transected, there is lossof 85% of the RGC bodies 2 weeks later.82,83 There is evi-dence that this death is apoptotic in nature,82 but the precisemechanism of axotomy-induced cell death remains unclear.RGC bodies have been noted to undergo apoptosis in glau-

Figure 5. Excitotoxicity is a component of ischaemic injury.Ischaemia leads to a depletion of adenosine triphosphate (ATP) andsubsequent loss of ionic homeostasis and depolarization. Uncon-trolled depolarization activates the self-reinforcing excitoxicitycascade.

IschaemiaNa+/K+ ATPasefailureATP

DepolarizationExcitotoxicitycascade



coma patients,84,85 a finding that is consistent with retrogradedegeneration after axonal injury, and consistent with thevascular, mechanical and target-derived trophic factortheories.

Evidence for excitotoxicity in the pathogenesis of glau-coma remains circumstantial and speculative, but is derivedfrom several sources: (i) evidence that vitreal glutamate levelsare elevated in experimental and clinical glaucoma; (ii) evi-dence that ischaemia plays a role in glaucoma; (iii) evidencefrom neuroprotection-based animal studies; and (iv) evi-dence of secondary degeneration of RGCs after optic nerveinjury. These lines of evidence will be discussed briefly inturn.

In 1996, Dreyer et al. reported that glutamate levels in thevitreous were increased in both experimental and clinicalglaucoma.86 This finding supported the concept that exces-sive glutamate levels and excitotoxicity were a componentof the pathogenesis of glaucoma.87 However, subsequentstudies by other investigators failed to corroborate this find-ing.88–90 Although the weight of evidence is against raisedvitreal glutamate levels in glaucoma, this does not mean thatextracellular retinal levels are not affected – they may beelevated, but this has not yet been proven.

Excitotoxicity is considered to be an important compo-nent of ischaemic injury (Fig. 4), as evidenced by the atten-uation of experimental ischaemic injury (to both retinal andoptic nerve head ischaemia) that is produced by glutamatereceptor blockade.91–94 Hence, if ischaemia plays a role in thepathogenesis of glaucoma then excitotoxicity may be a com-ponent. Given the large amount of evidence that optic nerveblood flow is compromised in glaucoma patients,71,95–97 andby inference, that the optic nerve head suffers ischaemicinjury, it follows that excitotoxic injury (as a component ofthe ischaemic injury) may be part of the pathogenesis. Thisline of reasoning is probably the strongest, although circum-stantial, evidence to support the notion that excitotoxicityis involved in the pathogenesis. However, the precise molec-ular mechanisms by which excitotoxicity would be a com-ponent of optic nerve head ischaemia remain unclear, anddifficulties with this concept will be discussed in the follow-ing text.

Neuroprotection-based studies not only provide informa-tion about potential management, but also provide informa-tion about pathogenesis: if an anti-excitotoxic drugattenuates injury in a particular model, then it follows thatexcitotoxicity is a component of the pathogenesis in thatmodel. NMDA receptor antagonists have been shown toattenuate RGC loss in rodent98 and primate models ofchronically elevated IOP;99 optic nerve ischaemia94 and afteroptic nerve crush injury in rodents.15,100 The ability ofNMDA receptor antagonists to attenuate injury is easiest toexplain when the injury involves ischaemia to a nerve cellbody or processes that contain NMDA receptors. For exam-ple, the ability of MK-801 (a well-studied blocker) to atten-uate RGC injury from pressure-induced retinal ischaemia isnot surprising.91 However, the mechanism by which NMDAreceptor blockade could attenuate ‘experimental glaucoma’

remains unclear. If glaucomatous pathology is principally atthe level of the unmyelinated axons at the optic nerve head,which have not been shown to have NMDA receptors, howcould NMDA blockade protect RGCs? Several explanationsfor the success of NMDA antagonists in these situationsexist: (i) the models inadvertently produce a primary injuryto the retina, and it is this injury that is being attenuated bythe NMDA blocker; (ii) NMDA receptors are present onunmyelinated optic nerve axons; (iii) NMDA inhibitionreduces injury to astrocytes and secondarily to axons; (iv)retrograde death of the RGC body and/or nerve fibre layerresults in release of glutamate into the extracellular spacewithin the inner retina and evokes the excitotoxicity cascadeand secondary degeneration of surrounding neurones; and(v) chronic injury to the optic nerve head causes a failure ofionic homeostasis, with a slow build-up of intracellular Na+

and depolarization of the axon and eventually the RGCsoma, leading to a ‘slow excitotoxicity’ situation as describedearlier.

Furthermore, there is evidence for secondary degenera-tion after optic nerve crush injury100 and optic nerve partialsection injury;101 however, the mechanism of the secondarydegeneration remains unclear. Also, it is very difficult inpractice to separate primary optic nerve degeneration fromsecondary degeneration. How does one know when primarydegeneration ends and secondary degeneration begins?Although RGC death may appear delayed, how can one besure that these cells have not suffered a ‘slow kill’. Yoles et al.described attenuation of RGC loss after optic nerve crushinjury by an NMDA antagonist.100 They proposed that thiswas evidence for secondary degeneration of RGCs unaf-fected by the initial insult. Theoretically, they are right: thisis evidence for secondary degeneration occurring at the levelof the inner retina, but it does not explain why cells that aredying by apoptosis (primary injury after optic crush) arereleasing glutamate and killing their neighbours. Spread ofapoptosis via gap junctions has been reported among hip-pocampal astrocytes.102 But whether or not apoptotic RGCsharm their neighbours, in particular via an excitotoxic mech-anism, remains unclear. Levkovitch-Verbin et al. in an elegantexperimental model have shown that after precise partialsection of the optic nerve, RGC bodies outside the regionof the axonotemesis are subsequently killed.101 However, itis unclear where the secondary degeneration is being initi-ated. The secondary degeneration may be initiated at thelevel of the white matter injury. Here, there would be bothaxonal cylinder and glial cell injury that could cause releaseof glutamate and initiate the excitotoxicity cascade (morelikely through AMPA/kainate than NMDA receptor activa-tion). Furthermore, given that oligodendrocytes service anumber of axons, it is possible that those injured at theborder zone caused demise of non-transected axons.

AMPA/kainate receptor antagonists have received rela-tively little attention as potential therapeutic agents for glau-coma; however, Schuettauf et al. have shown that inhibitionof these receptors after optic nerve crush attenuated RGCdeath more effectively than inhibition of NMDA receptors.15

60 Casson


POTENTIAL ANTI-EXCITOTOXIC DRUGS FOR THE TREATMENT OF GLAUCOMA

Brimonidine

Several ocular hypotensive agents that are currently used totreat glaucoma have been shown under laboratory condi-tions to have neuroprotective properties. Of these, brimoni-dine and betaxolol have the greatest supporting evidence.Brimonidine is generally considered to provide neuroprotec-tion via anti-apoptotic properties rather than direct inhibi-tion of ion channels.103,104 It has been shown to have noeffect on NMDA-stimulated Ca2+ entry.105 Hence, it is notan anti-excitotoxic agent per se, but can ameliorate down-stream intracellular effects of excess Ca2+ entry. Brimonidinehas been shown to attenuate RGC loss after optic nervecrush106 and laser-induced ocular hypertension in rodents.107

Brimonidine has recently been shown to improve contrastsensitivity in glaucoma patients and the weight of evidencein favour of brimonidine as a potential neuroprotectiveagent has led to a large, multicentre, prospective random-ized study comparing the effect of brimonidine to timololon the visual field changes in patients with normal tensionglaucoma.108

Betaxolol

In addition, to its primary action as a selective beta-blocker,betaxolol has other interesting pharmacological propertiesthat make it a candidate as an anti-excitotoxic agent. Thereis considerable laboratory evidence to indicate that betaxololhas calcium and sodium channel-blocking properties andthat it can attenuate ischaemic retinal injury.109–112 It has beenshown to inhibit NMDA-stimulated Ca2+ influx.105

There is some evidence to suggest that betaxolol providesbetter preservation of the visual field than timolol despiteless effect on the IOP.113,114 These intriguing results mayrelate to pressure-independent, neuroprotective effects;however, large-scale evidence is lacking.

Memantine

Memantine is classified as an uncompetitive NMDA antag-onist, binding near the Mg2+ site within the ion channel.115

An uncompetitive antagonist is defined as an inhibitor whoseaction is contingent upon prior activation of the receptor bythe agonist: the degree of blockade for a given concentrationof antagonist increases as the concentration of the agonistincreases.115 This is an ideal situation for an NMDA blocker:anti-excitotoxicity therapy needs to block excessive activa-tion of the NMDA receptor while leaving normal functionrelatively unscathed.

Memantine has recently been shown (in a randomized,placebo-controlled study) to be clinically effective in thetreatment of moderate to severe Alzheimer’s disease.68 Fur-thermore, memantine has recently been shown to providestructural protection to RGCs in a primate model of glau-

coma.116 In addition, the memantine-treated primate eyesshowed a relative sparing of the changes to the visuallyevoked cortical response and the multifocal electroretino-gram.99 A large-scale, randomized study is currently inprogress to determine the effect of memantine on visual fieldloss in glaucoma patients. The results are not expected until2007.

Neuroprotection-based human studies can provideinvaluable insights into the pathogenesis of glaucoma. If, forinstance, memantine does prove to be clinically useful, thenit will not only have provided the first accepted, pressure-independent treatment, but will have also proved that exci-totoxicity plays a role in the pathogenesis of the disease.

CONCLUSION

Excitotoxicity describes the process of neuronal injury byexcess stimulation of amino acid receptors. This form ofinsult was initially described in the retina, and subsequentlyhas been shown to be an important component of the patho-genesis of CNS injury. Although anti-excitotoxic treatmenthas proved unsuccessful in the treatment of stroke, there isincreasing evidence that excitotoxicity is involved in severalchronic neurological conditions, and anti-excitotoxic treat-ment has already been approved for some of these. There isexperimental evidence from animal models that excitotoxicinjury may play a role in glaucoma; however, extrapolatingfindings from the laboratory to the clinic is fraught withproblems, particularly when the pathogenesis of the diseaseis poorly understood. The results of the ‘Memantine Study’should be available soon. If this did demonstrate a protectiveeffect it would be an enormous advance in not only ourmanagement, but also our understanding of glaucoma. Itwould still leave many unanswered questions, but would bean ‘exciting’ first step.

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Possible role of excitotoxicity in the pathogenesis of glaucoma