**3. Animal models**

lase (PYGL), glutamate-ammonia ligase (GLUL) and glutamate dehydrogenase 1 (GLUD1). [38] Activation of these enzymes eventually stimulates the Krebs cycle and oxidative phos‐ phorylation, thereby increasing mitochondrial ROS production. Secondly, after TNF-α stim‐ ulation, RIP1 forms a complex with TNFR, Riboflavin kinase, and NADPH oxidase 1. [39, 40] NADPH oxidase is the best characterized non-mitochondrial source of ROS and forms a membrane bound enzyme complex with p22phox and Rac. [41] Thirdly, RIP1 kinase acti‐ vates autophagic degradation of catalase, which converts hydrogen peroxide to water and oxygen, thereby increasing ROS accumulation. [42] More recently, activation of the ne‐ crosome has shown to interact with the mixed lineage kinase domain-like (MLKL) and phosphoglycerate mutase 5 (PGAM5) resulting in the fusion of mitochondria and necrotic

A, In response to TNF-α stimulation, RIP1 is recruited to TNFR and forms a membrane associated complex I with TRADD, TRAF2/5 and cIAP1/2, which in turn leads to polyubiquitination of RIP1 and pro-survival NF-κB activation. B, RIP1 switches function to a regulator of cell death when RIP1 is unubiquitinated by A20 or CYLD. Deubiquitination of RIP1 leads to the formation of cytosolic DISC with FADD and caspase-8, the so-called complex II. In contrast to TNF signaling, Fas stimulation directly forms DISC. Activation of caspase-8 in DISC leads to apoptosis induction. During apoptosis, RIP1 is cleaved and inactivated by caspase-8. C, In conditions where caspases are blocked or cannot be acti‐ vated efficiently, RIP1 binds to RIP3, and both RIP1 and RIP3 kinases are phosphorylated at the RIP1-RIP3 complex. RIP1 kinase phosphorylation is critical for necrosis induction. In response to TNF-α, RIP1 binds to NADPH oxidase 1 and produces superoxide. Activated RIP3 binds to PYGL, GLUL and GLUD1 and increases the production of mitochondrial ROS. ROS overproduction leads to mitochondrial dysfunction, resulting in the release of mitochondrial pro-death pro‐ teins. Activation of the necrosome has been shown to interact with mixed lineage kinase domain-like (MLKL) and

phosphoglycerate mutase 5 (PGAM5) resulting in the fusion of mitochondria and necrotic cell death

cell death. [43, 44]

182 Glaucoma - Basic and Clinical Aspects

**Figure 2. Schematic of the RIPK signaling pathway.**

Animal experimental models in glaucoma research are produced by inducing either an ele‐ vation in intraocular pressure or damage to the axons of retinal ganglion cells. [77] Several methods have been employed to raise intraocular pressure in animal models. They range from obliteration of episcleral vessels [78] to iatrogenic sclerosis of the trabecular mesh‐ work (laser-induced [79] or through retroinjection of hypertonic saline into the limbal plexus [80]) or to mechanical obstruction of the trabecular meshwork with polystyrene beads. [81] Direct damage to retinal ganglion cell axons can be achieved via axotomy or crushing of axons. Retinal ganglion cell death occurs in 1-2 weeks from the time of optic nerve transection or crushing of axons in a fairly predictable fashion. [82] Interestingly, there is a specific mouse strain (DBA/J2) which inherently produces a much slower degen‐ eration of retinal ganglion cells; a process thought to more closely mimic human disease than other induced mouse models of glaucoma. [83] Mutations in the transmembrane gly‐ coprotein Gpnmb (premature stop codon at position 150 -GpnmbR150X) and the presence of the Tyrp1b gene allele (the b mutation is in a heme-associated domain and renders the protein susceptible to rapid proteolytic degradation) in the DBA/2J mouse model are thought to cause pigment dispersion and iris atrophy respectively. Both proteins are highly expressed in melanocytes and are involved in melanin and cell growth regulation. The de‐ creased levels of Gpnmb and Tyrp1 lead to cell death and abnormal melanin content re‐ lease that deposits onto the trabecular meshwork resulting in elevated IOP. [84] Of note, these mutations have not been shown to cause glaucoma in patients.

roprotective agents. [89] First, apoptosis of retinal ganglion cells has been inhibited in the lab through the use of anti-excitotoxic agents that primarily inhibit or interfere with the glu‐ tamate excitotoxic cascade. [90] Glutamate is a natural neurotransmitter that is required by the organism for proper cell signaling including retinal ganglion cells. Glutamate acts through many types of glutamate receptors/ion channels. One of these receptors/ion chan‐ nels is the *N*-methyl-D-aspartate (NMDA) type, which leads to calcium flux upon activation. Persistent activation of this channel by glutamate or NDMA leads to excitotoxicity. Gluta‐ mate induced excitotoxicity has been shown in some but not all animal models and elevated glutamate levels have been detected in some but not all studies (reviewed in [91]). Meman‐ tine is an uncompetitive antagonist of the *N*-methyl-D-aspartate (NMDA) type of glutamate receptor/channel. It can only bind to this ion channel in its "open" state, that is after gluta‐ mate has already bound to its receptor and has caused the channel to open. [92] Studies on several animal models of glaucoma have supported the neuroprotective role of memantine on retinal ganglion cells. [93]. Activated glutamate receptor leads to increased calcium flux. Calcium levels are important in many neuronal signaling events and aberrant calcium levels are thought to be important mediators of neuronal cell death. Increased levels of intracellu‐ lar calcium can be very detrimental to the health of the cell. Most recently, inhibitors of the L-type voltage-gated calcium channel, clinidipine [94] and lomerizine [95] as well as the al‐ pha-2 adrenergic agonist brimonidine [96] have also been shown to limit glutamate-induced excitotoxicity. Although the exact mechanism of action of brimonidine remains unknown, intraperitoneal pre-treatment with brimonidine has been shown by several groups to in‐ crease survival of retinal ganglion cells after optic nerve or retinal injury in animal models of

Neuroprotection in Glaucoma http://dx.doi.org/10.5772/54294 185

Antiapoptotic strategies utilize neurotrophins [101] or aim at the activation of Bcl-2 antia‐ poptotic pathways. [102] Neurotrophins are factors that signal the survival and growth of neurons. The first neurotrophin to be discovered was Nerve Growth Factor (NGF) in the 1960s. In 1986 Levi-Montalcini and Cohen shared the Nobel prize "for their discovery of growth factors for neurons." Neurotrophins are peptides that bind to cell surface receptors and activate survival signals, thus suppressing the apoptotic process (Fig. 4). [80, 103, 104] Exogenous administration of brain-derived neurotrophic factor (BDNF) or nerve growth fac‐ tor (NGF) has delayed but not prevented retinal ganglion cell death. [105-108] Injection of BDNF, ciliary neurotrophic factor (CNTF), neurotrophin-4 (NT-4), fibroblast growth factor-2 (FGF-2), and neurturin (a ligand for a glial cell line-derived neurotrophic factor family relat‐ ed receptor A2 - GFRA2) into the vitreous has also increased survival of retinal ganglion cells. [105, 109, 110] Neurotrophin delivery and overexpression via viral vector delivery seems to promote survival of retinal ganglion cells even further. [106] Finally, agents that interact with the two major neurotrophin cell surface receptor systems, tropomyosin-related kinase (Trk) receptor and p75 neurotrophin receptor (p75NTR), can also prolong survival of retinal neuronal tissue (Fig. 4). [111-113] Neurotrophins are difficult to be used in clinics be‐ cause of their polypeptidic nature: they are destroyed in the acidic milieu of the stomach, while their size hinders their ability of crossing the blood-brain barrier. Thus, special techni‐ ques have to be invented, such as intravitreal implants of cells producing these molecules

locally, such as the CNTF producing cells by Neurotech (Cumberland, RI, USA).

NDMA excitotoxicity, optic nerve crush and ischemia. [97-100]

The ideal animal model should manifest slow focal injury to retinal ganglion cell axons at the optic nerve head that leads to sectoral death of retinal ganglion cells without loss of other retinal neurons. [77] The currently used animal models for glaucoma research are far from ideal and their limitations are partly responsible for the failure to translate results from the bench to the bedside. For the elevated IOP models, it is clear that there are differ‐ ent susceptibilities of retinal ganglion cell damage among different species (mice, rats, monkeys) and among different strains or age of the same species. [51, 85, 86] For the axoto‐ my models, acute damage to retinal ganglion cell axons is different from the slow progres‐ sion seen clinically in glaucoma, and thus it remains unclear whether studies with these models can safely reproduce glaucomatous damage as it occurs in humans. [82] For the DBA/2J mouse, there is significant variability in glaucoma progression among animals and between eyes of the same animal, which renders any comparison study particularly diffi‐ cult. [77]

An inherent limitation of using animal models for the study of any human disease process is that animal models often lack the heterogeneity, compounded comorbidities, and polyphar‐ macy that are present in human pathologic conditions. Moreover, it is difficult to extrapolate from animal studies what the appropriate dose of a putative neuroprotective agent would be in human subjects since pre-clinical studies rarely assess for a dose-response curve, thera‐ peutic index, and central nervous system penetration. [87, 88]

### **4. Success in the lab**

Efforts in pre-clinical studies have targeted the various mechanisms that produce axonal de‐ generation and retinal ganglion cell death and have led to the discovery of an array of neu‐ roprotective agents. [89] First, apoptosis of retinal ganglion cells has been inhibited in the lab through the use of anti-excitotoxic agents that primarily inhibit or interfere with the glu‐ tamate excitotoxic cascade. [90] Glutamate is a natural neurotransmitter that is required by the organism for proper cell signaling including retinal ganglion cells. Glutamate acts through many types of glutamate receptors/ion channels. One of these receptors/ion chan‐ nels is the *N*-methyl-D-aspartate (NMDA) type, which leads to calcium flux upon activation. Persistent activation of this channel by glutamate or NDMA leads to excitotoxicity. Gluta‐ mate induced excitotoxicity has been shown in some but not all animal models and elevated glutamate levels have been detected in some but not all studies (reviewed in [91]). Meman‐ tine is an uncompetitive antagonist of the *N*-methyl-D-aspartate (NMDA) type of glutamate receptor/channel. It can only bind to this ion channel in its "open" state, that is after gluta‐ mate has already bound to its receptor and has caused the channel to open. [92] Studies on several animal models of glaucoma have supported the neuroprotective role of memantine on retinal ganglion cells. [93]. Activated glutamate receptor leads to increased calcium flux. Calcium levels are important in many neuronal signaling events and aberrant calcium levels are thought to be important mediators of neuronal cell death. Increased levels of intracellu‐ lar calcium can be very detrimental to the health of the cell. Most recently, inhibitors of the L-type voltage-gated calcium channel, clinidipine [94] and lomerizine [95] as well as the al‐ pha-2 adrenergic agonist brimonidine [96] have also been shown to limit glutamate-induced excitotoxicity. Although the exact mechanism of action of brimonidine remains unknown, intraperitoneal pre-treatment with brimonidine has been shown by several groups to in‐ crease survival of retinal ganglion cells after optic nerve or retinal injury in animal models of NDMA excitotoxicity, optic nerve crush and ischemia. [97-100]

methods have been employed to raise intraocular pressure in animal models. They range from obliteration of episcleral vessels [78] to iatrogenic sclerosis of the trabecular mesh‐ work (laser-induced [79] or through retroinjection of hypertonic saline into the limbal plexus [80]) or to mechanical obstruction of the trabecular meshwork with polystyrene beads. [81] Direct damage to retinal ganglion cell axons can be achieved via axotomy or crushing of axons. Retinal ganglion cell death occurs in 1-2 weeks from the time of optic nerve transection or crushing of axons in a fairly predictable fashion. [82] Interestingly, there is a specific mouse strain (DBA/J2) which inherently produces a much slower degen‐ eration of retinal ganglion cells; a process thought to more closely mimic human disease than other induced mouse models of glaucoma. [83] Mutations in the transmembrane gly‐ coprotein Gpnmb (premature stop codon at position 150 -GpnmbR150X) and the presence of the Tyrp1b gene allele (the b mutation is in a heme-associated domain and renders the protein susceptible to rapid proteolytic degradation) in the DBA/2J mouse model are thought to cause pigment dispersion and iris atrophy respectively. Both proteins are highly expressed in melanocytes and are involved in melanin and cell growth regulation. The de‐ creased levels of Gpnmb and Tyrp1 lead to cell death and abnormal melanin content re‐ lease that deposits onto the trabecular meshwork resulting in elevated IOP. [84] Of note,

The ideal animal model should manifest slow focal injury to retinal ganglion cell axons at the optic nerve head that leads to sectoral death of retinal ganglion cells without loss of other retinal neurons. [77] The currently used animal models for glaucoma research are far from ideal and their limitations are partly responsible for the failure to translate results from the bench to the bedside. For the elevated IOP models, it is clear that there are differ‐ ent susceptibilities of retinal ganglion cell damage among different species (mice, rats, monkeys) and among different strains or age of the same species. [51, 85, 86] For the axoto‐ my models, acute damage to retinal ganglion cell axons is different from the slow progres‐ sion seen clinically in glaucoma, and thus it remains unclear whether studies with these models can safely reproduce glaucomatous damage as it occurs in humans. [82] For the DBA/2J mouse, there is significant variability in glaucoma progression among animals and between eyes of the same animal, which renders any comparison study particularly diffi‐

An inherent limitation of using animal models for the study of any human disease process is that animal models often lack the heterogeneity, compounded comorbidities, and polyphar‐ macy that are present in human pathologic conditions. Moreover, it is difficult to extrapolate from animal studies what the appropriate dose of a putative neuroprotective agent would be in human subjects since pre-clinical studies rarely assess for a dose-response curve, thera‐

Efforts in pre-clinical studies have targeted the various mechanisms that produce axonal de‐ generation and retinal ganglion cell death and have led to the discovery of an array of neu‐

these mutations have not been shown to cause glaucoma in patients.

peutic index, and central nervous system penetration. [87, 88]

cult. [77]

**4. Success in the lab**

184 Glaucoma - Basic and Clinical Aspects

Antiapoptotic strategies utilize neurotrophins [101] or aim at the activation of Bcl-2 antia‐ poptotic pathways. [102] Neurotrophins are factors that signal the survival and growth of neurons. The first neurotrophin to be discovered was Nerve Growth Factor (NGF) in the 1960s. In 1986 Levi-Montalcini and Cohen shared the Nobel prize "for their discovery of growth factors for neurons." Neurotrophins are peptides that bind to cell surface receptors and activate survival signals, thus suppressing the apoptotic process (Fig. 4). [80, 103, 104] Exogenous administration of brain-derived neurotrophic factor (BDNF) or nerve growth fac‐ tor (NGF) has delayed but not prevented retinal ganglion cell death. [105-108] Injection of BDNF, ciliary neurotrophic factor (CNTF), neurotrophin-4 (NT-4), fibroblast growth factor-2 (FGF-2), and neurturin (a ligand for a glial cell line-derived neurotrophic factor family relat‐ ed receptor A2 - GFRA2) into the vitreous has also increased survival of retinal ganglion cells. [105, 109, 110] Neurotrophin delivery and overexpression via viral vector delivery seems to promote survival of retinal ganglion cells even further. [106] Finally, agents that interact with the two major neurotrophin cell surface receptor systems, tropomyosin-related kinase (Trk) receptor and p75 neurotrophin receptor (p75NTR), can also prolong survival of retinal neuronal tissue (Fig. 4). [111-113] Neurotrophins are difficult to be used in clinics be‐ cause of their polypeptidic nature: they are destroyed in the acidic milieu of the stomach, while their size hinders their ability of crossing the blood-brain barrier. Thus, special techni‐ ques have to be invented, such as intravitreal implants of cells producing these molecules locally, such as the CNTF producing cells by Neurotech (Cumberland, RI, USA).

RGCs. [67] Other studies have shown that treatment with minocycline reduces RGC death in experimental glaucoma. [127] The antiapoptotic effects of minocycline are not very clear and seem to be pleiotropic. They are exerted, at least in part, by modulating inflammation and metalloproteinases and by reducing mitochondrial calcium overloading. Minocycline stabilizes the mitochondrial membrane and inhibits release of cytochrome c and other apop‐ totic factors into the cytoplasm, thus resulting in decreased caspase activation and nuclear damage. Minocycline also exerts caspase-independent neuroprotective effects including up‐ regulation of anti-apoptotic factor Bcl-2. Another promising agent is Rasagiline, a monoa‐ mine oxidase inhibitor that has been found have neuroprotective and anti-apoptotic effects partially through increase in the mitochondrial family of Bcl-2 proteins, prevention in the fall in mitochondrial membrane potential, prevention of the activation of caspase 3, and of translocation of glyceraldehyde-3-phosphate dehydrogenase from the cytoplasm to the nu‐ cleus. It can also affect the secretion of amyloid precursor protein (APP) and it has been shown to delay RGC cell death in experimental glaucoma [128]. However, it has to be noted that it has not taken approval by the United States Food and Drug Administration in a Par‐ kinson's disease trial. Erythropoietin (EPO) activates the NF-κB pathway and results in prosurvival and anti-oxidant enzyme upregulation and has been shown to be protective of RGC in the DBA/2J mouse of pigmentary glaucoma [129]. Citicholine (cytidine 5'-diphosphocho‐ line) which exhibits neuroprotective effects by preserving cardiolipin and sphingomyelin among other actions, has been tested in patients with anterior ischemic optic neuropathy

Neuroprotection in Glaucoma http://dx.doi.org/10.5772/54294 187

Over the last 30 years, numerous pharmacologic agents and gene therapeutic approaches have been shown to be neuroprotective in animal models of retinal and optic nerve injury. To date, none of the trials on neuroprotection of the visual system have shown efficacy and none of the agents developed in the laboratory have translated into a definitive clinical treat‐ ment. [131] There are several reasons for the universal failure of clinical trials to confirm preclinical results; they stem from the nature of glaucoma itself and from the poor design of previous neuroprotective trials. [51, 131] First, the long, slow course of glaucomatous optic neuropathy hinders our efforts to measure whether an improvement in progressive worsen‐ ing has been achieved and asks for a design of therapeutic trials that last several years. Sec‐ ond, the rate of worsening varies among patients and thus a larger sample size is required to account for this inherent variability in disease progression. Third, the current standard of outcome measurement remains visual field testing and this carries a significant test-retest variance even among patients who are adept in taking the test. Fourth, any neuroprotection clinical trial would have to include patients that are already on topical medications that low‐ er IOP. IOP-lowering agents have proven effective in slowing glaucoma progression in sev‐ eral controlled clinical trials [8-10] and it would be unethical to preclude neuroprotection

and showed preliminary benefits. [130]

**5. Difficulties in designing a clinical trial**

study patients from using IOP-lowering medications.

**Figure 4.** Neurotrophin Signaling Summary. (courtesy of Dr A. Gravanis). Neurotrophins include the first to be discov‐ ered: Nerve Growth Factor (NGF) as well as Brain Derived Neurotrophic Factor (BDNF) and Neurotrophin 3 and 4 (NT-3 and NT-4). There are two classes of receptors for neurotrophins: p75 and the "Trk" family of Tyrosine kinase receptors. P75 can bind all factors, whereas Trk receptors are specific for their ligands. Binding of neurotrophins to their recep‐ tors leads to activation of many pro-survival signals including PI3K, Akt, and MAPK among others.

Tumor necrosis factor-alpha (TNF-α) can lead to death of retinal ganglion cells by inducing mitochondrial damage and activation of caspases. [114] Inhibition of TNF-α via the use of etanercept [115] or an anti-TNF-α neutralizing antibody [116] can protect retinal ganglion cells in mouse models of glaucoma.

Providing free radical scavengers, such as coenzyme Q10 [117, 118] or thioredoxin [119], or in‐ hibiting the formation of free radicals by blocking the action of nitric oxide synthase [120] has shown promise as an alternative neuroprotective strategy. Inflammatory and immune mecha‐ nisms also play a role in glaucomatous damage of retinal ganglion cells. [121] Immunomodula‐ tion [122], inhibition of calcineurin by tacrolimus [123], and subcutaneous injection of granulocyte-colony stimulating factor [124] also have neuroprotective potential in glaucoma.

Other mechanisms that have been investigated in the lab with encouraging results include the use of mesenchymal stem cells, which are thought to exert their effect through produc‐ tion of neurotrophins or stimulation of inflammation [125, 126]. A recent study has shown that amyloid β (Aβ) is found to be elevated in an animal model of ocular hypertension in‐ duced glaucoma and that inhibition of the generation of amyloid β led to preservation of RGCs. [67] Other studies have shown that treatment with minocycline reduces RGC death in experimental glaucoma. [127] The antiapoptotic effects of minocycline are not very clear and seem to be pleiotropic. They are exerted, at least in part, by modulating inflammation and metalloproteinases and by reducing mitochondrial calcium overloading. Minocycline stabilizes the mitochondrial membrane and inhibits release of cytochrome c and other apop‐ totic factors into the cytoplasm, thus resulting in decreased caspase activation and nuclear damage. Minocycline also exerts caspase-independent neuroprotective effects including up‐ regulation of anti-apoptotic factor Bcl-2. Another promising agent is Rasagiline, a monoa‐ mine oxidase inhibitor that has been found have neuroprotective and anti-apoptotic effects partially through increase in the mitochondrial family of Bcl-2 proteins, prevention in the fall in mitochondrial membrane potential, prevention of the activation of caspase 3, and of translocation of glyceraldehyde-3-phosphate dehydrogenase from the cytoplasm to the nu‐ cleus. It can also affect the secretion of amyloid precursor protein (APP) and it has been shown to delay RGC cell death in experimental glaucoma [128]. However, it has to be noted that it has not taken approval by the United States Food and Drug Administration in a Par‐ kinson's disease trial. Erythropoietin (EPO) activates the NF-κB pathway and results in prosurvival and anti-oxidant enzyme upregulation and has been shown to be protective of RGC in the DBA/2J mouse of pigmentary glaucoma [129]. Citicholine (cytidine 5'-diphosphocho‐ line) which exhibits neuroprotective effects by preserving cardiolipin and sphingomyelin among other actions, has been tested in patients with anterior ischemic optic neuropathy and showed preliminary benefits. [130]
