**13. Immune modulation**

#### **13.1. Anti-inflammatory agents**

There is an inflammatory component to the neuronal retinal degeneration in glaucoma [75-78]. Studies have proved an age related susceptibility of glaucoma victims to progressive nerve damage and RGC loss even with single digit IOP [4]. Researchers have also established elements of the complement pathway such as C1q, as markers for astrocyte destruction that may result in RGC apoptosis [78]. Thus, the future of glaucoma therapy lies in employing additional modalities based on proven mechanisms of RGC loss.

stimulating caspase ultimately leading to RGC death [79] (Figure 1). However the binding of the TNF-R1 receptor also triggers via heat shock proteins and activation of transcription factor NF-KB, a cell survival pathway. TNF- α levels if at best optimized (kept low) create a homeo‐ stasis that facilitates a balance between neuroprotection and neurodegeneration [75,79].

Strategies for Neuroprotection in Glaucoma http://dx.doi.org/10.5772/53776 215

TNF- α is one of a 19-member family of ligands that exert their inflammatory activities through 29 receptors, triggering a cascade of inflammatory responses. TNF- α has been found to be upregulated in neurodegenerative diseases such as parkinson's and alzheimer's disease. In studies on the brain tissue of Alzheimer's patients, TNF- α, a mediator of chronic inflammation,

TNF- α in some studies was found in high concentration after laser-induced OHT, along with increased macrophage/microglia near the optic nerve head [77]. The levels of TNF α surpassed those in other inflammatory processes not involving ocular hypertension, validating the protein as a likely mediator of the RGC death, and hence a target for gene therapy. The increase in microglia population in the vitreous surface around the optic nerve head also alludes to an inflammation theory of glaucomatous optic nerve and RGC damage [77]. Research in alz‐ heimer's has established TNF-α as a mediator of chronic inflammation with detection of increased levels in the brain of victims of this neurodegenerative disorder [76]. Serum amyloid A, is another acute-phase inflammatory marker discovered in the retina and trabecular meshwork of glaucoma eyes [78]. An understanding of the molecular processes in parallel

Agarwal et al 2012, reported on extensive research evidence in support of the role of TNF-α in glaucomatous optic nerve degeneration and RGC apoptosis [75]. Research involved eyes with POAG, NTG and exfoliative glaucoma, with cataract eyes as controls [75]. Results showed marked elevation in TNF- α in aqueous samples of all glaucoma groups compared with controls. In addition, optic nerve degeneration and RGC loss were demonstrated in eyes

Under normal conditions there is greater expression of TNF-R1 over TNF- α. Stress factors such as trauma, ischaemia, and elevated hydrostatic pressure result in an increase in expression of TNF-R1 and TNF- α. These have been shown to have several roles including pro-apoptotic and neuroprotective properties depending on the environment in which they are expressed [Figure 3]. Experimental evidence using mouse eyes have shown that in the absence of normal glial cells, the apoptotic effect dominates. Microglial cells are thought to provide survival signals necessary for the neuroprotective effect of TNF- α. Insults such as ischemia, oxidative stress and optic nerve injury increases the expression of cell death signals and reduces the expression of the cell survival signals, thereby potentiating the harmful effects of TNF- α [61].

In contrast, normally functioning glial cells support the neuroprotective effects of TNF- α and TNF-R1. The ischaemic and hydrostatic stress in glaucoma activate microglial activity causing an inflammatory response. Activated glial cells produce TNF- α along with harmful com‐ pounds like NO and endothelin 1 (ET-1). In excessive microglial activation, up regulation of

disorders will influence the outlook on the future of glaucoma management.

has been detected in increased levels [76].

subject to intravitreal injection of TNF- α.

*13.2.1. How does TNF- α work?*

**Figure 3.** Apoptosis Pathway for Retinal Ganglion Cells

#### **13.2. Tumour Necrosis Factor α (TNF α)**

Progression of optic nerve axonal degeneration and retinal ganglion cell (RGC) apoptosis, have been shown to be responsible for progressive visual field loss in glaucoma, with or without ocular hypertension. One mechanism has been linked to tumor necrosis factor (TNF-α) in tissue around the optic nerve head demonstrated during immunostaining of mouse specimen [77]. This protein is a pro-inflammatory cytokine produced in response to trauma and inflammation and can start the apoptotic cascade [77] (Figure 3). TNF-α, secreted by damaged glials cells and through the binding of TNF receptor-1 (TNF-R1) starts the apoptotic process stimulating caspase ultimately leading to RGC death [79] (Figure 1). However the binding of the TNF-R1 receptor also triggers via heat shock proteins and activation of transcription factor NF-KB, a cell survival pathway. TNF- α levels if at best optimized (kept low) create a homeo‐ stasis that facilitates a balance between neuroprotection and neurodegeneration [75,79].

TNF- α is one of a 19-member family of ligands that exert their inflammatory activities through 29 receptors, triggering a cascade of inflammatory responses. TNF- α has been found to be upregulated in neurodegenerative diseases such as parkinson's and alzheimer's disease. In studies on the brain tissue of Alzheimer's patients, TNF- α, a mediator of chronic inflammation, has been detected in increased levels [76].

TNF- α in some studies was found in high concentration after laser-induced OHT, along with increased macrophage/microglia near the optic nerve head [77]. The levels of TNF α surpassed those in other inflammatory processes not involving ocular hypertension, validating the protein as a likely mediator of the RGC death, and hence a target for gene therapy. The increase in microglia population in the vitreous surface around the optic nerve head also alludes to an inflammation theory of glaucomatous optic nerve and RGC damage [77]. Research in alz‐ heimer's has established TNF-α as a mediator of chronic inflammation with detection of increased levels in the brain of victims of this neurodegenerative disorder [76]. Serum amyloid A, is another acute-phase inflammatory marker discovered in the retina and trabecular meshwork of glaucoma eyes [78]. An understanding of the molecular processes in parallel disorders will influence the outlook on the future of glaucoma management.

Agarwal et al 2012, reported on extensive research evidence in support of the role of TNF-α in glaucomatous optic nerve degeneration and RGC apoptosis [75]. Research involved eyes with POAG, NTG and exfoliative glaucoma, with cataract eyes as controls [75]. Results showed marked elevation in TNF- α in aqueous samples of all glaucoma groups compared with controls. In addition, optic nerve degeneration and RGC loss were demonstrated in eyes subject to intravitreal injection of TNF- α.

#### *13.2.1. How does TNF- α work?*

may result in RGC apoptosis [78]. Thus, the future of glaucoma therapy lies in employing

Progression of optic nerve axonal degeneration and retinal ganglion cell (RGC) apoptosis, have been shown to be responsible for progressive visual field loss in glaucoma, with or without ocular hypertension. One mechanism has been linked to tumor necrosis factor (TNF-α) in tissue around the optic nerve head demonstrated during immunostaining of mouse specimen [77]. This protein is a pro-inflammatory cytokine produced in response to trauma and inflammation and can start the apoptotic cascade [77] (Figure 3). TNF-α, secreted by damaged glials cells and through the binding of TNF receptor-1 (TNF-R1) starts the apoptotic process

additional modalities based on proven mechanisms of RGC loss.

214 Glaucoma - Basic and Clinical Aspects

**Figure 3.** Apoptosis Pathway for Retinal Ganglion Cells

**13.2. Tumour Necrosis Factor α (TNF α)**

Under normal conditions there is greater expression of TNF-R1 over TNF- α. Stress factors such as trauma, ischaemia, and elevated hydrostatic pressure result in an increase in expression of TNF-R1 and TNF- α. These have been shown to have several roles including pro-apoptotic and neuroprotective properties depending on the environment in which they are expressed [Figure 3]. Experimental evidence using mouse eyes have shown that in the absence of normal glial cells, the apoptotic effect dominates. Microglial cells are thought to provide survival signals necessary for the neuroprotective effect of TNF- α. Insults such as ischemia, oxidative stress and optic nerve injury increases the expression of cell death signals and reduces the expression of the cell survival signals, thereby potentiating the harmful effects of TNF- α [61].

In contrast, normally functioning glial cells support the neuroprotective effects of TNF- α and TNF-R1. The ischaemic and hydrostatic stress in glaucoma activate microglial activity causing an inflammatory response. Activated glial cells produce TNF- α along with harmful com‐ pounds like NO and endothelin 1 (ET-1). In excessive microglial activation, up regulation of TNF α - causes RGC apoptosis in the absence of normal glial support. If there is significant microglial insult early in the event, TNF α - continues to exert apoptosis even after the stimulus is removed, as has been shown in in-vivo studies with mice, where progression of RGC death was seen on immunostaining even after normal IOPs were reached [75].

*13.5.1. Opioids*

**14. Stem cells**

**15. Gene therapy**

nerve head (neuroprotection) [4].

Opioid receptor activation has been shown to reduce the ischemic damage to the retina as demonstrated by ERG [84]. Opioid receptor stimulation and the facilitation of the actions of endogenous opioids show promise in neuroprotection of RGCs in glaucoma [84,85]. In the mice model, glaucoma was induced by raising the IOP above the systolic blood pressure (155-160mmHg) for 45 minutes to induce ischemic retinal injury [84]. The opioid antagonist naloxone (3mg/kg) was given to mice intraperitoneally 24 hours before the ischemic event. Another study group of mice had morphine (0.01-10mg/kg given intraperitoneallly 24 hours before the ischemic injury. 7 days after the injury the retina of both groups were assessed by the ERG. The mice that has morphine had greater preservation of their ERG a and b wave amplitudes 7 days after the ischemic event. Further the protective effect of morphine on preservation of ERG amplitudes was dose related with the ED50 of 0.18mg/kg [84]. However, these strategies have not yet been tested in humans or undergone randomized controlled trials.

Strategies for Neuroprotection in Glaucoma http://dx.doi.org/10.5772/53776 217

Much research is still yet to be done on stem cells and neuroprotection. Stem cells can supply neurotrophins and modulate matrix metalloproteinases after an injury which can be neuro‐ protective and limit neuronal damage [86]. However in a pre-clinical model of glaucoma, intravitreal stem cell injections have been shown to enhance the survival of the RGC [23].

With emerging evidence for the molecular basis in glaucoma- pathophysiology, the disease may be interrupted by targeting key sites once the genetic expression is known. Studies of micro-RNA such as miRNA-125b has led to the understanding of the key sites for targeted down-regulation of messenger RNA which is thought to add to the oxidative stress induction of inflammation and astrogliosis in alzheimer's disease [78]. Alzheimer's disease, parkinson and glaucoma are thought to have a similar neurodegenerative basis (molecular and cellular pathways for neuronal cell loss) [78]. Hence gene therapy for glaucoma and other neurode‐ generative disorders may be where medical management is headed. Target sites include uveoscleral outflow site, surgical (trabeculectomy) site, ciliary apparatus, retina and optic

Gene therapy would be helpful in preventing neurodegeneration using anti apoptotic genes, bcl-2 and bcl-x [2]. Another mechanism is blocking the apoptotic pathway with deprenyl (monamine oxidase inhibitor). It is proposed that it stabilizes the mitochondrial membrane potential, preventing the release of cytochrome c which can activate capsases (Figure 1) [2, 87].

Targeting antioxidant genes is a promising strategy for future management of glaucomatous neurodegeneration. Researchers used cloned extracellular superoxide dismutase (ECSOD) or

#### **13.3. Agmatine, an aminoguanidine**

Current research targets TNF-α for neuroprotection by reducing RGC loss (Figure 3). Such agents need to have high selectivity and specificity for excessive TNF-α and TNF-1 expression while preserving local immunity. Agents such as Agmatine, an aminoguanidine, have been shown to protect RGCs against the apoptotic effects of TNF-α, but the effects on other receptors and pathways are yet to be established [44,45,75]. Agmatine has been used at a concentration of 60 mg daily in rat ocular hypertension model [31]. 10(-3) M agmatine solution 4 times a day has shown a high affinity for alpha 2 receptors on the ciliary body, where it exerts its IOP lowering effect which has been seen in the rat model [80]. Amnioguanidine also targets inducible nitric oxide synthase (iNOS) inhibitors [31,73].

#### **13.4. Ethanrecept, the future in neuroprotection**

Work done by Roh et. al (2012) demonstrated the ability of ethanrecept, a recombinant chimeric protein, to act as a TNFα inhibitor to reduce RGC loss in the wake of elevated TNFα [77]. This decoy protein selectively binds TNFα, sparing the RGC damage from this and other inflam‐ matory agents such as microglia [77]. Ethanrecept is used in the treatment of juvenile idiopathic arthritis, rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis, and has shown no IOP-lowering capabilities. The drug however shows promise as a neuroprotective agent for intravitreal use in the future.

#### **13.5. Copolymer -1 (Cop-1), — A possible vaccination for neuroprotection?**

The inflammatory process in neurodegenerative diseases such as alzheimer's and glaucoma has been found to be associated with pro-inflammatory activities mediated in part by T cell activity. Cop-1, a synthetic peptide polymer known to suppress autoimmune encephalomye‐ litis, modulates this T cell reaction by producing a Th2 anti-inflammatory phenotype with attenuation of normal inflammatory response in neurodegenerative diseases as well as increased neuroprotection [13, 81,82]. Cop-1, glatiramer acetate has been FDA approved in the treatment of multiple sclerosis, a demyelinating disease.

It had been noted experimentally that an eye that had recent glutamate injections had resulting large numbers of lymphocytes present, hence it was theorized that glutamate toxicity induces a T cell lymphocyte reaction [81]. Therefore, by immunizing against this with the correct antigen, theoretically could reduce the damage induced by the glutamate. Cop-1 immunization has shown some protection against glutamate toxicity and elevated IOP in mice retinal ganglion cells [81,82,83]. So T cell mediated immunoprotection may be a future option for glaucoma, however, much research is still to be done.

#### *13.5.1. Opioids*

TNF α - causes RGC apoptosis in the absence of normal glial support. If there is significant microglial insult early in the event, TNF α - continues to exert apoptosis even after the stimulus is removed, as has been shown in in-vivo studies with mice, where progression of RGC death

Current research targets TNF-α for neuroprotection by reducing RGC loss (Figure 3). Such agents need to have high selectivity and specificity for excessive TNF-α and TNF-1 expression while preserving local immunity. Agents such as Agmatine, an aminoguanidine, have been shown to protect RGCs against the apoptotic effects of TNF-α, but the effects on other receptors and pathways are yet to be established [44,45,75]. Agmatine has been used at a concentration of 60 mg daily in rat ocular hypertension model [31]. 10(-3) M agmatine solution 4 times a day has shown a high affinity for alpha 2 receptors on the ciliary body, where it exerts its IOP lowering effect which has been seen in the rat model [80]. Amnioguanidine also targets

Work done by Roh et. al (2012) demonstrated the ability of ethanrecept, a recombinant chimeric protein, to act as a TNFα inhibitor to reduce RGC loss in the wake of elevated TNFα [77]. This decoy protein selectively binds TNFα, sparing the RGC damage from this and other inflam‐ matory agents such as microglia [77]. Ethanrecept is used in the treatment of juvenile idiopathic arthritis, rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis, and has shown no IOP-lowering capabilities. The drug however shows promise as a neuroprotective agent

The inflammatory process in neurodegenerative diseases such as alzheimer's and glaucoma has been found to be associated with pro-inflammatory activities mediated in part by T cell activity. Cop-1, a synthetic peptide polymer known to suppress autoimmune encephalomye‐ litis, modulates this T cell reaction by producing a Th2 anti-inflammatory phenotype with attenuation of normal inflammatory response in neurodegenerative diseases as well as increased neuroprotection [13, 81,82]. Cop-1, glatiramer acetate has been FDA approved in the

It had been noted experimentally that an eye that had recent glutamate injections had resulting large numbers of lymphocytes present, hence it was theorized that glutamate toxicity induces a T cell lymphocyte reaction [81]. Therefore, by immunizing against this with the correct antigen, theoretically could reduce the damage induced by the glutamate. Cop-1 immunization has shown some protection against glutamate toxicity and elevated IOP in mice retinal ganglion cells [81,82,83]. So T cell mediated immunoprotection may be a future option for

**13.5. Copolymer -1 (Cop-1), — A possible vaccination for neuroprotection?**

was seen on immunostaining even after normal IOPs were reached [75].

inducible nitric oxide synthase (iNOS) inhibitors [31,73].

treatment of multiple sclerosis, a demyelinating disease.

glaucoma, however, much research is still to be done.

**13.4. Ethanrecept, the future in neuroprotection**

for intravitreal use in the future.

**13.3. Agmatine, an aminoguanidine**

216 Glaucoma - Basic and Clinical Aspects

Opioid receptor activation has been shown to reduce the ischemic damage to the retina as demonstrated by ERG [84]. Opioid receptor stimulation and the facilitation of the actions of endogenous opioids show promise in neuroprotection of RGCs in glaucoma [84,85]. In the mice model, glaucoma was induced by raising the IOP above the systolic blood pressure (155-160mmHg) for 45 minutes to induce ischemic retinal injury [84]. The opioid antagonist naloxone (3mg/kg) was given to mice intraperitoneally 24 hours before the ischemic event. Another study group of mice had morphine (0.01-10mg/kg given intraperitoneallly 24 hours before the ischemic injury. 7 days after the injury the retina of both groups were assessed by the ERG. The mice that has morphine had greater preservation of their ERG a and b wave amplitudes 7 days after the ischemic event. Further the protective effect of morphine on preservation of ERG amplitudes was dose related with the ED50 of 0.18mg/kg [84]. However, these strategies have not yet been tested in humans or undergone randomized controlled trials.
