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Abstract: Neuroprotection in the field of glaucoma is defined as any treatment, independent of IOP reduction, which prevents RGC death. Glutamate antagonists, ginkgo biloba extract, neurotrophic factors, antioxidants, calcium channel blockers, brimonidine, glaucoma medications with blood regulatory effect and nitric oxide synthase inhibitors are among compounds with possible neuroprotective activity in preclinical studies. A few agents (such as brimonidine or memantine) with neuroprotective effects in experimental studies have advanced to clinical trials.
Nevertheless, lack of compelling clinical evidence has not prevented the off‑label use of some of these compounds in glaucoma practice. Stem cell transplantation has been reported to halt experimental neurodegenerative disease processes in the absence of cell replacement. The advantage of this approach is a prolonged and targeted effect. Neuroprotection in glaucoma, pharmacologically or by stem cell transplantation, is an interesting subject waiting for broad and multidisciplinary collaborative studies to better clarify its role in clinical practice.
Objective: This article is a review to understand the pathophysiology of RGC damage and potential role of neuroprotective agents in glaucoma .This review, highlights some of the ocular pharmacological approaches that are being used and/or tested to reduce neurodegeneration and provide some form of neuroprotection.
Methodology: This systematic review is based on an extensive literature search of publications in PubMed, Google Scholar and Cochrane. The keywords used for the literature search included glaucoma, stem cells, Brimonidine; Glaucoma; Memantine; Neuroprotection; Retinal ganglion cells , Stem Cell therapy and therapeutics. Inclusion criteria were English language publications, all study designs, and publications from 2000 to 2018. A comparative evaluation of the results of each study was also made.Meta- analysis could not be done due to the heterogeneity of settings, interventions and outcomes. A synthesis of the included studies was performed.
Introduction
Glaucoma is a multifactorial disease with well-described risk factors such as IOP, age, race, family history and myopia.[15]. The exact mechanism(s) of RGC damage in glaucoma is complex and unknown. Glaucoma represents share certain clinical characteristics, including excavation of the optic disc and loss of the RGC with resultant visual field loss, with or without elevated IOP.
Globally, glaucoma is the most common optic neuropathy, the second most common cause of blindness and the most common cause of preventable visual disability.[1] It encompasses a spectrum of progressive optic neuropathies characterized by pathological degeneration of nonmyelinated retinal ganglion cells (RGCs), with structural damage at the optic nerve head. Irrespective of the multitude of potential initiating insults, the common theme in the pathogenesis of glaucoma is the triggering of a cascade that results in accelerated apoptosis of the RGCs.[2–4] This process of cellular death occurs in the absence of inflammation, and is characterized by DNA fragmentation, chromosome clumping, cell shrinkage and membrane blebbing. As a consequence of neuronal death within the central visual pathway, clinical signs of glaucoma include retinal nerve fiber layer defects, neuroretinal rim thinning with excavation of the optic nerve head ('cupping') and irreversible visual field loss. Neural degeneration in glaucoma is not limited to the retina; it also affects neurons in the lateral geniculate nucleus and visual cortex.[5,6]
Although intraocular pressure (IOP) is no longer part of the definition of glaucoma, it is the most easily modifiable risk factor to decrease both risk of disease onset and disease progression. IOP reduction by medical, laser surgical means remains the only clinically proven treatment for glaucoma,[7–9] but it is not entirely effective for all patients, as exemplified in the Collaborative Normal Tension Glaucoma Study: despite IOP reduction, glaucomatous optic degeneration continued, albeit more slowly and in a smaller proportion of patients.[8] Sufficient IOP reduction to arrest the disease process entirely may be difficult, or may provoke significant adverse side effects. These inadequacies in current treatment paradigm have prompted research into neuroprotection as an alternative strategy for glaucoma. The processes of killing neurons in glaucoma: hypoxia, trophic insufficiency, oxidative stress, excitotoxicity, immune related attack and apoptotic death. Agents that benefit these neurodegenerative disorders may also assist in glaucoma.[10–12]Summary of causes of retinal ganglion cell injury and potential sites for neuroprotection shown in Table 1.
Mechanisms of RGC Damage
S2-Evidence indicates that apoptosis may be the final common pathway for RGC death in glaucoma. Apoptosis is a programmed cell death pathway that occurs without eliciting an inflammatory response.. Markers of apoptosis have also been observed in the human glaucomatous retina [15.16,]. Several mechanisms that may initiate RGC apoptosis in glaucoma have been proposed (Fig. 1). These include neurotrophic factor deprivation, hypoperfusion/ischemia, glial cell activation, glutamate excitotoxicity, and abnormal immune response.
Table 1. Summary of causes of retinal ganglion cell injury and potential sites for neuroprotection.
BDNF: Brain-derived neurotrophic factor; CNTF: Ciliary neurotrophic factor; NMDA: N-methyl-D- aspartate; RGC: Retinal ganglion cell.
Neurotrophic Factors deprivation
A major destructive effect of increased or fluctuating IOP is deformation of the lamina cribrosa, mechanically compressing RGC axons. This reduces or blocks retrograde transport of essential neurotrophic factors such as brain-derived neurotrophic factor (BDNF), NGF, neurotrophin (NT)-3, NT-4 and NT-5, glial cell-derived neurotrophic factor, ciliary neurotrophic factor, and FGF-2, liberated by the superior colliculus and lateral geniculate body and transported to the RGC body by its axons.[17–19]
Ischemia
Another major theory in the etiology of glaucoma is vascular insufficiency at the optic nerve head.[19,20] Arising from systemic hypotension, vasospasm or even mechanical compression of the microvasculature at the lamina cribrosa, low perfusion of the optic nerve head may cause RGC ischemia. This ischemic insult may reduce essential nutrients and substrates available for energy production in metabolically highly active neurons. Antivasospastic drugs such as calcium-channel blockers and some adrenergic antagonists have potential as neuroprotectants.
Mitochondrial Dysfunction
Increasingly, mitochondrial dysfunction is believed to contribute to the pathogenesis of neurodegenerative disorders, including glaucoma.[22]. Mitochondrial dysfunction induces the intrinsic apoptotic pathway by upregulation of NF-κB and proapoptotic genes. As mitochondrial dysfunction may be triggered by aging, ischemia and/or oxidative stress, novel methods such as caloric restriction (to try to retard aging[23]), increasing optic head flow dynamics (with vasodilators) and decreasing oxidative stress (with antioxidants) may prove to be useful neuroprotective strategies.
Glutamate Excitotoxicity
Any hypoxic environment critically drops ATP production with failure of the vital sodium–potassium pump of both neurons and their supporting glia. excessive levels of glutamate are toxic not only to RGCs, but also to neighbouring healthy neurons causes calcium influx through hyperactivation of the N-methyl- D-aspartate (NMDA) receptor in a process termed excitotoxicity. Overstimulation of NMDA receptors also activates nitric oxide synthase (NOS), resulting in nitric oxide (NO) production. NO is a neuronal messenger critical for normal retinal neurotransmission and phototransduction. Unregulated, it has the potential to react with the superoxide anion to form peroxynitrite, a highly reactive oxidant species.[24]
Oxidative Stress
A number of investigations have supported the role of oxidative stress in the pathogenesis of glaucoma.[25] These mainly demonstrated lower levels of antioxidants[26,27] and elevated oxidative stress markers in the aqueous humor of eyes with glaucoma,[27] antibodies against glutathione‑S‑transferase,[28] decreased plasma levels of glutathione[29] and increased lipid peroxidation products in the plasma of glaucoma patients.[30] Furthermore, tissue analysis studies comparing cultured human trabecular meshwork (TM) from eyes with POAG to that of non glaucomatous eyes have revealed higher concentrations of reactive oxygen species, decreased cell membrane potentials and reduced ATP production in the TM of eyes with POAG.[31] .Oxidative free radicals have been implicated in human TM degeneration and subsequent IOP increase and glaucoma.[35].
Misfolded proteins
Misfolded proteins such as amyloid β (Aβ) are a prominent feature of many neurodegenerative diseases, including Alzheimer's, Huntington's and Parkinson's, with an accumulation of abnormal protein plaques in the brain. As Aβ has been linked to glaucomatous RGC apoptosis in a dose- and time-dependent manner,[30] targeting different components of the Aβ formation and aggregation pathways (e.g., using Aβ antibodies) may effectively reduce glaucomatous RGC apoptosis.[31]
Glial Cell Modulation
Retinal ganglion cells are not the only cells damaged in glaucoma: Müller glial cells, amacrine and bipolar cells are also injured. In the nonmyelinated region of the optic nerve head, astrocytes are the major glial cells to provide support to neuronal axons, as well as interface between connective tissue and blood vessels. To try to maintain homeostasis, quiescent astrocytes are transformed into a reactive state by liberated cytokines such as TGF,[34] ciliary neurotrophic factor,[35] FGF[36] and PDGF.[37] Reactive astrocytes exhibit altered intercellular communication, migration, growth factor signaling, oxidative species buffering capacity and connective tissue properties at the optic nerve head.[38]
Apoptotic Death Pathways
The final common pathway for any neuronal injury is necrosis or apoptosis, the latter playing a major role in RGC death in glaucoma. Apoptosis can be initiated by extrinsic or intrinsic pathways. Triggers for the extrinsic pathway include TNF-α, Fas ligand and TNF-relatedapoptosis-inducing ligand. The intrinsic pathway involves mitochondrial-mediated events. The exact processes of apoptosis and neuronal cell death are well described.[41] Regardless of the initiating injury, there is activation of the caspase cascade,[42] increased expression of proapoptotic genes such as Bax/Bid[43] and downregulation of antiapoptotic genes such as Bcl-2/Bcl-xl,[44] leading to noninflammatory programmed cell death.[45] Taking a lead from viruses that use caspase inhibition to prevent apoptosis of infected cells, pharmacological interventions that block the caspase cascade may be neuroprotective.
Figure 1. Simplified pathway of RGC death and assumed mechanisms of neuroprotective agents. IOP, intraocular pressure; NMDA, n-methyl-D- aspartate; NOS, nitric oxide synthase; RGC, retinal ganglion cell.
One of the areas of great interest in glaucoma is how RGC death occurs.[46] The molecular basis of RGC death stems from investigations on animal models of glaucoma. Deprivation of neurotrophic factors,[47] elevated concentrations of excitatory aminoacids such as glutamate,[48] and oxidative stress[49] may contribute to RGC apoptosis [Figure 2].
Neuroprotective Compounds In The Treatment Of Glaucoma
IOP reduction per se can prevent or delay RCG death in glaucomas and therefore is indirectly neuroprotective. However, neuroprotection in glaucoma is defined as any intervention, independent of IOP reduction, that can prevent RGC death. Several natural and synthetic compounds, have been reported to possess neuroprotective properties. Neuroprotection can affect glaucoma by direct protection of RGCs or neutralization of the deleterious effects of toxic factors. The present article reviews current evidence on neuroprotective compounds in the treatment of glaucoma.
Drugs with dual pharmacophoric activities
At present two recently FDA-approved novel drugs, namely netarsudil 0.02% [52] and latanoprostene bunod 0.024% [53]. Netarsudil inhibits rho kinase and norepinephrine transporter it relaxes the TM and Schlemm’s canal (SC) cells (thereby helping aquous humor (AQH) to drain via the conventional pathway), and it inhibits Na+/K +-ATPase in the ciliary epithelial cells thereby inhibiting AQH production and lowering IOP. In a similar vein, latanoprostene bunod releases latanoprost free acid (LFA) and nitric oxide (NO) the FP-receptors in ciliary muscle and TM are activated by LFA to cause local release of MMPs that digest extracellular matrix (ECM) to create/enlarge the UVS outflow pathway and promote AQH drainage from both the UVS and TM/SC pathways, while the NO activates soluble guanylate cyclase in TM/SC cells (Dismuke et al., 2009, 2010) that produces cGMP that relaxes TM/SC cells and enhances conventional outflow of AQH. Indeed, such studies have been conducted in POAG/OHT patients and the results are encouraging for this novel formulation containing both netarsudil and latanoprost [54].
Figure 2. Proposed mechanism leading to retinal ganglion cell death in glaucoma. MMP, matrix metalloproteinase. Ophthalmol Clin N Am 18 (2005) 383 – 395
Ginkgo Biloba Extract
Ginkgo is an ancient species of tree similar to plants which were living 270 million years ago. This tree is widely grown in China and was introduced early in traditional Eastern medicine to treat a variety of problems such as asthma, vertigo, fatigue and tinnitus or circulatory disorders. In modern medical science, the extract from the leaves of ginkgo biloba, named as ginkgo biloba extract 761 (EGb761), has been shown to be beneficial for cognitive impairment and dementia.[43]
This is the only antioxidants capable of penetrating into the mitochondria can be of benefit as neuroprotective agents. Ginkgo contains certain substances, including polyphenolic flavonoids which may theoretically prevent oxidative stress in the mitochondria and thereby protect RGCs.[46-48]
In a crossover randomized clinical trial, ginkgo biloba extract (GBE) improve significantly in visual field indices in NTG patients. Some glaucomatologists have been prescribing GBE for their patients as adjuvant therapy for several years.[52] However, increasing risk of bleeding during surgery has been a cause of concern in patients using ginkgo.[53] Efficacy and safety reports have recommended a daily dose of 120 mg of GBE.[51]
Neurotrophic Factors
Among a variety of candidate growth and trophic factors for RGCs, brainderived neurotrophic factor (BDNF), appears to be of particular importance to RGC function and survival.[60‑64] BDNF has been shown to undergo both anterograde and retrograde axonal transport,[65] Quigley et al suggested the optimal dose of BDNF to be 0.01 mg per milliliter of vitreous volume for intravitreal injections and found that higher intravitreal doses decrease the protective effect of BDNF on RGCs possibly due to down regulation of Trk B, the BDNF receptor.[56]
Another trophic factor undergoing preclinical investigation is the human ciliary neurotrophic factor (CNTF). Pease et al assessed virally mediated over expression of CNTF and BDNF in an experimental model. Loss of RGC axons was 15% lower in CNTF treated retinas than in controls; however, neither the combined CNTF‑BDNF group nor the BDNF over expression group showed any significant improvement in RGC survival.[63] Artemin,[64] basic fibroblast growth factor,[65] interleukin‑6[66] and erythropoietin[67] are other trophic factors or cytokines for which a neuroprotective effect has been proposed.
Purified Recombinant Trophic Factors
The blood retina barrier impedes such large proteins from reaching the retina with systemic administration. Intravitreal injection is an alternative route to deliver purified recombinant trophic factors to the retina. Valproate, phenytoin; anti inflammatory agents like ibudilast, aspirin and meloxicam) could be synthesized, formulated and delivered intravitreally to slow down the death of RGCs and their axons. [64]
Alpha 2 Adrenergic Agonists Including Brimonidine
The presence of alpha adrenergic receptors in human, bovine and porcine retinas, particularly in RGCs and the inner nuclear layer has been demonstrated.[69,70]
It has been suggested that brimonidine may prevent RGC death by direct interaction with alpha‑2 adrenergic receptors, leading to reduced accumulation of extracellular glutamate and blockade of NMDA receptors; this protective effect is thought to be independent of IOP reducing mechanisms attributed to this agent.[71‑73] Elimination of the protective effect of brimonidine by coadministration of an alpha 2 antagonist confirms that the mentioned effect is secondary to alpha‑2 receptor activation.[71,74].
In another study, brimonidine treatment also preserved morphology, density and the total number of axons in the optic nerve subjected to high IOP.[75]
Nitric Oxide Synthase Inhibitors
Evidence in the literature points to a possible role for NO in RGC degeneration.[76‑78].There are three forms of nitric oxide synthase (NOS): NOS‑1 (neuronal NOS) and NOS‑3 (constitutive NOS) act as vasodilators or neurotransmitters in normal retinal tissue, however NOS‑2 (inducible NOS) contributes to RGC neurotoxicity.[80] An increased expression of NOS has been shown in optic nerve head (ONH) of glaucoma patients.[81,82].The possibility that NOS ‑2 inhibition could be neuroprotective in glaucoma was strengthened by reports showing that another NOS‑2 inhibitor (N‑nitro‑L‑arginine) delayed RGC degeneration.[83] The non‑psychotropic component of marijuana, cannabidiol (CBD), and the synthetic cannabinoids, tetrahydrocannabinol and HU‑211 have been demonstrated to possess protective actions in part due to an effect on reducing formation of lipid peroxides, nitrite/nitrate and nitrotyrosine.[86,84,85] These data suggest that activation of NOS, especially NOS‑2, may play a significant role in glaucomatous optic neuropathy and that nitric oxide synthase inhibitors could halt neurodegeneration.
Calcium Channel Blockers
The neurotoxic effect of NMDA is mediated by calcium influx into neural cells, followed by apoptosis and cell death.[86] Thus, calcium channel blockers (CCBs) seem to be a rational alternative for neuroprotection in glaucoma. CCBs theoretically rescue RGCs by prevention of cell death mediated by calcium influx and by improving local blood flow in ischemic tissues by inducing vasodilation.[87]
Different calcium channel blockers such as iganidipine, nimodipine and lomerizine have been shown to significantly increase purified rat RGC viability under hypoxia.[88].The effect of 2% topical flunarizine reduced IOP and attenuated injury to the retina, including RGCs.[82]
Other members of this family, brovincamine and nilvadipine, have high blood brain barrier permeability and are expected to induce favorable effects in the optic nerve or retina with minimal influence on systemic blood pressure.[83] They were shown to improve visual field defects and ocular circulation in NTG patients and diminished the rate of deterioration in visual field sensitivity of NTG patients in randomized clinical trials.[91‑93]
Antioxidants
Theoretically, inhibition of ROS and upregulation of cell defense systems may enhance RGC survival.[94-97] Cell defense mechanisms against oxidative stress include the superoxide dismutase, glutathione (GSH) and thioredoxin (TRX) systems.[95] The TRX system mitigates oxidative damage by scavenging intracellular ROS. The reaction leads to TRX oxidation, which is returned to its reduced form by TRX reductase in the presence of NADPH.
Anti‑Glaucoma Medications With Blood Regulation Effect
Vascular dysregulation has been implicated in the pathogenesis of glaucoma,[98]; therefore, a neuroprotective effect has been suggested for agents which can improve regulation of ocular blood perfusion.[99] Some antiglaucoma medications have additional ocular blood perfusion effects. For instance, carbonic anhydrase inhibitors increase ocular perfusion.[100] Improvement of ocular blood has also been reported with latanoprost.[101,102]. Betaxolol is a putative selective B1‑adrenoceptor blocker.[103,104 ]. Some studies have suggested that betaxolol reduces the NMDA stimulated influx of calcium into isolated cells of rat retinas by direct interaction with voltage‑dependent calcium channels or sodium channels.[105]
Stem Cell Transplantation For Rgc Neuroprotection
A systematic review to determine whether stem cell therapy had the potential to treat glaucoma. Nine studies were selected based on the predetermined inclusion and exclusion criteria. Of these nine studies, eight focused on neuroprotection conferred by stem cells, and the remaining one on neuroregeneration. Results from these studies showed that there was a potential in stem cell based therapy in treating glaucoma, especially regarding neuroprotection via neurotrophic factors. The studies revealed that a brain-derived neurotrophic factor expressed by stem cells promoted the survival of retinal ganglion cells in murine glaucoma models. The transplanted cells survived without any side effects. While these studies proved that stem cells provided neuroprotection in glaucoma, improvement of vision could not be determined. Clinical studies would be required to determine whether the protection of RGC correlated with improvement in visual function. Furthermore, these murine studies could not be translated into clinical therapy due to the heterogeneity of the experimental methods and the use of different cell lines. In conclusion, the use of stem cells in the clinical therapy of glaucoma will be an important step in the future as it will transform present-day treatment with the hope of restoring sight to patients with glaucoma.[106]
Summary
Over the past 30 years, numerous pharmacologic agents have been advocated as neuroprotective agents in glaucoma, however few of them such as brimonidine or memantine have advanced to clinical trials.
Glaucoma is a chronic heterogenous group of disorders, and no animal model can fully mimic the course of human disease. Furthermore, considerable disease variability exists in human clinical trials; these include the presence of comorbidities, polypharmacy in elderly glaucoma patients, and minimal control over a myriad of physiologic factors.
Successful clinical application of one or more neuroprotective strategies depends on several factors: (1) the strategy has to have a rational scientific basis; (2) the neuroprotective agent must be delivered safely and efficiently to the site of damage; and (3) the efficacy and safety profile of the neuroprotective agent must be demonstrated in a randomized prospective clinical trial. For a chronic, slowly progressive disease such as glaucoma, proving clinical efficacy remains a challenge because it may take many years to detect significant benefit. Nonetheless, the goal of clinically significant optic nerve protection in glaucoma seems within reach.
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Author: Dr. Kazi Reshad Agaz
MS(Ophth), FICO(UK), MCPS (Ophth), DO, MBBS.
Consultant Ophthalmologist, Bangladesh Eye Hospital and Institute 78, Satmasjid Road, Dhaka
