Advances in Neuroprotection in Glaucoma: Pharmacological Strategies and Emerging Technologies

Abstract

Glaucoma is a major global health concern and the leading cause of irreversible blindness worldwide, characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons. This review focuses on the need for neuroprotective strategies in glaucoma management, addressing the limitations of current treatments that primarily target intraocular pressure (IOP) reduction. Despite effective IOP management, many patients continue to experience RGC degeneration, leading to irreversible blindness. This review provides an overview of both pharmacological interventions and emerging technologies aimed at directly protecting RGCs and the optic nerve, independent of IOP reduction. Pharmacological agents such as brimonidine, neurotrophic factors, memantine, Ginkgo biloba extract, citicoline, nicotinamide, insulin, and resveratrol show promise in preclinical and early clinical studies for their neuroprotective properties. Emerging technologies, including stem cell therapy, gene therapy, mitochondrial-targeted therapies, and nanotechnologies, offer innovative approaches for neuroprotection and regeneration of damaged RGCs. While these interventions hold significant potential, further research and clinical trials are necessary to confirm their efficacy and establish their role in clinical practice. This review highlights the multifaceted nature of neuroprotection in glaucoma, aiming to guide future research and clinical practice toward more effective management of glaucoma-induced neurodegeneration.

1. Introduction

Glaucoma is a major global health concern and is recognized as one of the primary causes of irreversible blindness worldwide. The condition is typified by the gradual deterioration of retinal ganglion cells (RGCs) and their axons, resulting in the loss of the visual field and ultimately irreversible blindness. With over 70 million individuals affected globally, the impact of glaucoma is substantial and is predicted to increase to 111.8 million affected individuals by 2040 [1].

While current treatments primarily focus on lowering intraocular pressure (IOP), RGC loss can occur even with normal IOP levels, and a significant proportion of patients continue to experience vision loss despite effective IOP management. There is increasing evidence that other mechanisms, such as oxidative stress, excitotoxicity, vascular dysregulation, and neuroinflammation, also play a significant role in the pathogenesis of the disease [2,3,4]. This has led to a growing interest in neuroprotective strategies that aim to directly preserve and protect RGCs and the optic nerve from neurodegenerative processes, independent of reducing IOP, therefore slowing down the decline in visual function. By focusing on neuroprotection, researchers hope to develop therapies that can halt or even reverse the damage to the optic nerve, providing a significant advancement in the management of glaucoma.

This review aims to provide a comprehensive overview of the current state of neuroprotective strategies for glaucoma, focusing on both pharmacological treatments and emerging technologies. By examining a wide range of approaches, this review seeks to highlight the multifaceted nature of neuroprotection. This holistic perspective is essential for understanding the potential benefits and limitations of each strategy, guiding future research and clinical practice towards a more effective management of glaucoma-induced neurodegeneration.

2. Mechanisms of Loss of Retinal Functions in Glaucoma

The key mechanisms underlying RGC death in glaucoma involve a complex interplay of various pathological processes, including neurotrophin deprivation, excitotoxicity, oxidative stress, mitochondrial dysfunction, inflammation, and apoptosis (Figure 1).

Neurotrophins, such as ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF), are crucial in the survival and maintenance of RGCs. In glaucoma, the deprivation of these neurotrophins due to impaired axonal transport and receptor expression leads to RGC degeneration [6].

Excitotoxicity, primarily driven by elevated levels of the neurotransmitter glutamate, leads to the overactivation of N-methyl-D-aspartate (NMDA) receptors on RGCs, causing a harmful influx of calcium ions that triggers apoptotic pathways. While the role of glutamate in glaucoma is complicated and not entirely understood, it is clear that its dysregulation contributes significantly to RGC death [2].

Oxidative stress is another critical factor in glaucomatous neurodegeneration. An imbalance between reactive oxygen species (ROS) production and the body’s antioxidant defenses leads to oxidative damage in retinal ganglion cells (RGCs), affecting proteins, lipids, and DNA. This oxidative stress can be exacerbated by mitochondrial dysfunction, as RGCs have high energy demands, and any impairment in mitochondrial function can lead to energy deficits, further compromising cell viability. The release of cytochrome c from damaged mitochondria into the cytoplasm is a key event that activates the apoptotic machinery in RGCs [3].

Inflammation also plays a significant role in glaucoma, with pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) being upregulated in the retina and optic nerve head. These cytokines can induce RGC apoptosis either directly or by exacerbating excitotoxicity and oxidative stress. Additionally, the glial cells in the retina, including astrocytes, Müller cells, and microglia, contribute to both protective and detrimental responses in glaucoma. While they can help clear debris and produce neurotrophic factors, their overactivation can lead to chronic inflammation and further RGC damage [4].

Apoptosis, or programmed cell death, is the final common pathway through which many of these mechanisms converge to cause RGC loss in glaucoma. Both intrinsic and extrinsic apoptotic pathways are activated in response to the various stressors affecting RGCs. Key molecules in these pathways include the Bcl-2 family of proteins, which control mitochondrial membrane permeability, and caspases, which are the executioners of apoptosis [7].

3. Pharmacological Interventions (Summary in Table 1)

3.1. Brimonidine

Brimonidine, an alpha-2 adrenergic agonist, widely used to reduce IOP, has demonstrated notable neuroprotective properties in the treatment of glaucoma. While its IOP-lowering effects are well documented, emerging evidence highlights its potential to protect RGCs through various mechanisms independent of IOP reduction.

Table 1. Summary of neuroprotective compounds for glaucoma with clinical trials.

Active Compound	Mechanism of Action	Route of Administration	Status in Clinical Studies	Clinical Trials
Brimonidine	Binds alpha-2 adrenergic receptors, regulates apoptotic proteins, upregulates neurotrophic factors, reduces NMDA 1 receptor excitotoxicity	Topical	FDA-approved for IOP 2 reduction, neuroprotective effects under investigation	Krupin et al., 2011 [8]; De Moraes et al., 2012 [9]
Neurotrophic Factors (BDNF 3, CNTF 4, NGF 5)	Binds to specific receptors (TrkB and TrkA), promotes survival of neurons, enhances axon regeneration, modulates apoptosis	Implants (CTNF), topical (rhNGF 6)	Ongoing Phase II trials (CTNF), Phase Ib trial showed safety (rhNGF)	Goldberg et al., 2023 [10]; Beykin et al., 2022 [11]
Memantine	Non-competitive NMDA receptor antagonist, reduces calcium influx, protects neurons from glutamate-induced excitotoxicity	Oral	Failed large-scale clinical trials	Weinreb et al., 2018 [12]
GBE 7	Scavenges ROS, reduces oxidative stress, stabilizes mitochondria, antagonizes PAF	Oral	Mixed results in clinical trials	Quaranta et al., 2003 [13]; Lee et al., 2013 [14]; Guo et al., 2014 [15]
Citicoline	Serves as precursor for phospholipids, enhances neurotransmitter synthesis	Oral, topical	Ongoing large-scale clinical trials	NCT05315206 NCT05710198
Nicotinamide	Replenishes NAD levels, supports mitochondrial function, regulates calcium homeostasis, reduces oxidative stress	Oral	Ongoing large-scale long-term trials	NCT05275738 NCT05405868

¹ N-methyl-D-aspartate, ² intraocular pressure, ³ brain-derived neurotrophic factor, ⁴ ciliary neurotrophic factor, ⁵ nerve growth factor, ⁶ recombinant human nerve growth factor, ⁷ ginkgo biloba extract.

Table 1. Summary of neuroprotective compounds for glaucoma with clinical trials.

Active Compound	Mechanism of Action	Route of Administration	Status in Clinical Studies	Clinical Trials
Brimonidine	Binds alpha-2 adrenergic receptors, regulates apoptotic proteins, upregulates neurotrophic factors, reduces NMDA 1 receptor excitotoxicity	Topical	FDA-approved for IOP 2 reduction, neuroprotective effects under investigation	Krupin et al., 2011 [8]; De Moraes et al., 2012 [9]
Neurotrophic Factors (BDNF 3, CNTF 4, NGF 5)	Binds to specific receptors (TrkB and TrkA), promotes survival of neurons, enhances axon regeneration, modulates apoptosis	Implants (CTNF), topical (rhNGF 6)	Ongoing Phase II trials (CTNF), Phase Ib trial showed safety (rhNGF)	Goldberg et al., 2023 [10]; Beykin et al., 2022 [11]
Memantine	Non-competitive NMDA receptor antagonist, reduces calcium influx, protects neurons from glutamate-induced excitotoxicity	Oral	Failed large-scale clinical trials	Weinreb et al., 2018 [12]
GBE 7	Scavenges ROS, reduces oxidative stress, stabilizes mitochondria, antagonizes PAF	Oral	Mixed results in clinical trials	Quaranta et al., 2003 [13]; Lee et al., 2013 [14]; Guo et al., 2014 [15]
Citicoline	Serves as precursor for phospholipids, enhances neurotransmitter synthesis	Oral, topical	Ongoing large-scale clinical trials	NCT05315206 NCT05710198
Nicotinamide	Replenishes NAD levels, supports mitochondrial function, regulates calcium homeostasis, reduces oxidative stress	Oral	Ongoing large-scale long-term trials	NCT05275738 NCT05405868

¹ N-methyl-D-aspartate, ² intraocular pressure, ³ brain-derived neurotrophic factor, ⁴ ciliary neurotrophic factor, ⁵ nerve growth factor, ⁶ recombinant human nerve growth factor, ⁷ ginkgo biloba extract.

Brimonidine has a quinoxaline chemical structure with an imidazoline ring that allows it to bind to alpha-2 adrenergic receptors in the eye. Several preclinical studies have explored the neuroprotective mechanisms of brimonidine. It has been shown to enhance the survival of RGCs and protect RGCs from various types of optic nerve injuries, including NMDA-induced neurotoxicity, ischemia, optic neuritis, and ocular hypertension [16,17,18,19,20,21,22,23]. It achieves these protective effects by regulating anti- or pro-apoptotic proteins, such as Bcl-2/BxL or Bax, and upregulating neurotrophic factors like brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), and their receptors [24,25,26]. Brimonidine also alleviates neuronal death by altering NMDA receptors and reducing glutamate accumulation post-injury, thereby decreasing excitotoxicity [27,28,29]. Additionally, brimonidine interferes with the amyloid-β pathway, reducing amyloid-β levels, which are implicated in RGC death [30,31]. Improvement in impaired vascular autoregulation, protection of the retina from ischemic injury, and support of neuronal regeneration were also observed [16,19,20,22,23,24,32,33,34]. These effects suggest that brimonidine’s neuroprotection is not solely reliant on its ability to lower IOP. Recent studies have also shown additive protective effects when combining brimonidine and ripasudil. This combined treatment modulates multiple signaling pathways, including the suppression of proinflammatory mediators and the increase in trophic factors, leading to enhanced RGC survival compared to single-agent treatments [35,36].

Clinical studies have provided mixed but promising results regarding the neuroprotective effects of brimonidine. The Low-pressure Glaucoma Treatment Study (LoGTS) compared the effects of brimonidine and timolol in patients with low-pressure glaucoma. Although both medications were similarly effective in lowering IOP, visual field deterioration was notably less frequent in the brimonidine group compared to the timolol group. However, caution is advised when interpreting the results due to the high dropout rate, especially among those taking brimonidine [8,9]. Topical brimonidine also enhanced contrast sensitivity regardless of its effects on IOP, whereas timolol showed no such improvement [37]. Another study suggests that brimonidine may preserve retinal nerve fiber layer (RNFL) independent of its IOP-lowering effects [38].

Despite these findings, the evidence for brimonidine’s neuroprotective effects in clinical settings remains inconclusive. The high attrition rates and adverse effects reported in some studies raise concerns about the consistency and applicability of these results. Large double-blinded, randomized controlled trials are needed to definitively determine the role of brimonidine in the treatment of glaucoma [39].

3.2. Neurotrophic Factors

Neurotrophic factors (NTFs) are essential molecules involved in neuron development, survival, and repair. They bind to specific receptors, activating tyrosine kinase signaling pathways, which lead to various neuroprotective actions, such as the promotion of axon regeneration and enhancement of neuronal cell function [40,41,42].

Neurotrophic factors such as nerve growth factor (NGF), BDNF, CNTF, FGF-2, glial cell-line-derived neurotrophic factor (GDNF), neurturin (NRTN), and neuritin are particularly relevant to glaucoma. These factors have been shown to prevent RGC death in several rodent models of glaucoma [43,44,45,46,47,48,49,50].

BDNF plays a crucial role in the survival of retinal ganglion cells (RGCs) by preventing apoptosis through the activation of extracellular signal-regulated kinases (Erk1/2) and c-jun, while inhibiting caspase 2 via its receptor, TrkB [51,52]. Research has shown significantly reduced levels of BDNF in the serum and aqueous humor of patients with normal tension glaucoma (NTG) and primary open-angle glaucoma (POAG) [44,53,54]. BDNF has also been shown to improve RGC survival and prevent RGC death induced by amyloid-β-triggered apoptosis in rat models [40,55,56]. Nonetheless, further studies are needed to clarify the causal link between BDNF and glaucoma and to assess the effectiveness of BDNF supplementation as a neuroprotective treatment.

CNTF is produced locally by RGCs, and its levels are also found to be decreased in the aqueous humor and lacrimal fluid of patients with POAG [57]. CNTF exerts its effects through a heterotrimeric receptor complex that includes CNTF receptor α, gp130, and leukemia inhibitory factor receptor β [58]. It can protect damaged RGCs directly by binding to receptors on their surface, or indirectly by influencing glial cell activity via the JAK/STAT, PI3K/AKT, and MAPK signaling pathways [59,60,61,62]. Preclinical studies have shown a promising neuroprotective ability of CNTF with single intravitreal injection [63]. The combination of CNTF with other drugs such as cyclic adenosine monophosphate (cAMP) and Rho kinase inhibitor was also investigated, with benefits shown in axon regeneration [64,65,66]. The NT-501 device, a polymer capsule housing a genetically modified human cell line that releases CNTF, has been designed for long-term delivery. Ongoing clinical trials are evaluating the therapeutic effectiveness of NT-501-encapsulated cell therapy in the treatment of glaucoma. Phase I trials showed that NT-501 implants were safe, and ongoing Phase II trials aim to evaluate the efficacy of improving visual fields and retinal structure [10,67,68,69].

NGF promotes the survival and regeneration of retinal and brain cells [70]. It works by interacting with TrkA and p75 neurotrophin receptors (p75NTR). When NGF binds to TrkA, it activates the PI3K/Akt pathway, which boosts Bcl-2 levels and lowers Bax proteins, ultimately inhibiting apoptosis [71]. It also activates the RAS and PLC pathways, which are involved in regulating cell survival and differentiation [72]. Topical application of NGF preserved RGCs and their axons in a model of ocular hypertension [43,73]. It has been shown that in advanced glaucoma patients, topical treatment with NGF improved optic nerve functions, including visual acuity, visual field, and contrast sensitivity [43]. In preclinical studies, recombinant human NGF (rhNGF) enhanced neuroprotection, neuroregeneration, and axon growth were associated with reduced p75NTR and increased TrkA phosphorylation [71,74,75,76]. RhNGF has been evaluated in clinical trials, showing safety in healthy volunteers and some neuroprotective effects in glaucoma patients [11,77].

Despite promising preclinical results, translating these findings into clinical practice poses several challenges. The effective and long-lasting delivery of NTFs to the retina is a significant hurdle. Intravitreal injections, though effective, may not be practical for chronic conditions like glaucoma that require long-term treatment. Future research should focus on long-term studies and innovative delivery systems to fully harness the potential of neurotrophic factors in glaucoma therapy.

3.3. Memantine

Memantine is a voltage-dependent, non-competitive NMDA receptor antagonist with low affinity that selectively binds to activated glutamatergic receptors [78,79], reducing excessive glutamatergic activity, calcium influx, and the activation of pro-apoptotic cascades without affecting normal neurotransmission [80,81], thereby protecting RGCs from excitotoxic damage. Its rigid tricyclic amine structure (1-amino-3,5-dimethyladamantane) allows it to effectively block the NMDA receptor channel by interacting with critical asparagine residues (N-sites) like N616 in the NR1 and NR2 subunits. This binding inhibits excessive calcium influx while preserving normal receptor activity. Key amino acids involved in memantine’s channel blockade include N616, A645, and Y647 in NR1, and N615 in NR2B.

In animal models, memantine has demonstrated promising neuroprotective effects. Intraperitoneal, subcutaneous, and oral administrations of memantine in monkeys and rats improve RGC survival, prevent optic nerve fiber loss, and maintain retinal and visual pathway function without adverse effects on normal eyes [80,82,83]. However, the role of elevated glutamate in glaucoma remains debated and inconsistent [84,85,86].

Although preclinical studies have shown promising results, large-scale clinical trials have not been able to confirm significant benefits of memantine in the treatment of glaucoma. Two double-masked, placebo-controlled, multicenter Phase III clinical trials (NCT00141882 and NCT00168350) involving 2296 patients with POAG over four years did not show that memantine delayed visual field progression compared to the placebo [12].

To better evaluate memantine’s potential in glaucoma treatment, future studies should consider including patients with early-stage glaucoma and a less heterogeneous population regarding progression risk factors. Longer study durations, higher doses, and alternative methods of medication delivery should be explored. Additionally, using more advanced imaging techniques could provide more objective structural parameters for assessing early glaucoma progression [12,87].

3.4. Ginkgo Biloba Extract

Ginkgo biloba has been used in traditional Chinese medicine for hundreds of years. Modern research has explored the neuroprotective properties of Ginkgo biloba extract (GBE), particularly in the context of neurodegenerative diseases like glaucoma [88,89].

GBE contains active compounds such as flavonoid glycosides and terpene lactones, including ginkgolides A, B, C, and bilobalide, which contribute to its neuroprotective effects. The flavonoid glycosides, with their polyphenolic structure with two aromatic rings and glycosidic attachments, exhibit antioxidant properties by scavenging ROS and reducing oxidative stress. Terpene lactones, particularly ginkgolide B with its cage-like structure, antagonize the platelet activator factor (PAF), reducing trauma-induced apoptosis and enhancing recovery from ischemic injury. Bilobalide, another key terpene with its trilactone rings, stabilizes mitochondria, further mitigating ROS production. GBE’s ability to reduce lipid peroxidation helps protect cellular membranes and enhance erythrocyte deformability and blood perfusion. Additionally, both flavonoids and terpenoids in GBE contribute to vasodilation, supporting improved blood flow in tissues, and further reinforcing its neuroprotective properties [90].

In preclinical studies, GBE has demonstrated significant neuroprotective effects, highlighting GBE’s potential to enhance RGC survival and protect against glaucomatous damage [91,92,93]. Clinical trials investigating the effects of GBE on glaucoma patients have yielded mixed results. One study noted improvements in preexisting visual field defects in patients with NTG after one month of taking GBE oral capsules [13]. Another 4-year long-term study found that taking GBE significantly slowed the progression of visual field defects in NTG patients without affecting IOP [14]. However, a randomized controlled trial (RCT) showed no effect on visual field performance or contrast sensitivity in NTG patients of the same GBE dosage and duration as the first study [15], highlighting the variability in clinical outcomes. Numerous studies have shown increased blood flow velocity and decreased vascular resistance in the retrobulbar and peripapillary vascular systems [94,95].

Despite promising preclinical findings, the clinical evidence for GBE’s neuroprotective effects in glaucoma remains inconclusive [90]. The discrepancies in clinical trial results underscore the need for further research to identify the specific conditions and patient populations that may benefit from GBE treatment. Future studies should consider enrolling patients with early-stage glaucoma, controlling for hypotensive treatments, and utilizing advanced imaging techniques. In summary, GBE holds promise as a neuroprotective treatment for glaucoma, but larger, well-designed clinical trials are needed to validate its effectiveness and determine its place in clinical practice.

3.5. Citicoline

Citicoline, also known as cytidine 5′-diphosphocholine, a naturally occurring compound, has garnered attention for its potential neuroprotective effects in the context of glaucoma.

The chemical structure of citicoline consists of cytidine linked to a diphosphate bridge and choline, allowing it to serve as a precursor for the synthesis of important phospholipids such as phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and cardiolipin, which are essential components of neuronal cell membranes [96]. These phospholipids are integral for the formation of synaptic membranes, enhancing the release and recycling of neurotransmitters like acetylcholine, dopamine, serotonin, and norepinephrine, which are crucial for normal neuronal function [97]. The choline component also facilitates the synthesis of acetylcholine. While stabilizing neuronal membranes and enhancing important neurotransmitter synthesis, citicoline reduces glutamate excitotoxicity, improves axonal transport deficits, and lowers oxidative stress by producing glutathione [98].

In animal models, citicoline has demonstrated significant neuroprotective effects. Studies have shown that the administration of citicoline can attenuate the decrease in RGC density and improve visual acuity and the integrity of the pre-chiasmatic white matter of the optic nerve without impacting IOP, indicating a neuroprotective effect [99].

Early studies demonstrated that intramuscular (IM) citicoline improved visual field performance in patients with POAG, with long-term benefits in visual field preservation [100]. Other studies have also shown improvements in visual evoked potentials (VEP) and pattern electroretinograms (PERG) with IM citicoline [101]. Studies evaluating the effects of oral citicoline reported similar benefits on retinocortical function [102,103]. Longitudinal studies have also shown that oral citicoline can slow the progression of glaucomatous damage, with significant improvements in RNFL and ganglion cell complex thickness [104]. The topical administration of citicoline eyedrops has also shown similar effects on retinocortical function, as measured by PERG and VEP [105,106,107,108], but with more adverse effects compared to oral formulation [109]. Other studies investigating the combination therapy of citicoline with drugs including docosahexaenoic acid, homotaurine, and vitamin E also showed significant visual improvement [110,111]. Citicoline-loaded liposomes were also investigated, showing promising results for improved bioavailability and sustained drug release in ocular applications [112].

The neuroprotective potential of citicoline in glaucoma has sparked several ongoing clinical trials to explore its therapeutic benefits. One such trial is a randomized controlled trial evaluating the effects of oral citicoline over a year in patients with OAG (NCT05315206). Furthermore, a large Phase III randomized controlled trial is being planned to determine the efficacy of citicoline eyedrops in slowing visual field deterioration and structural changes in glaucoma patients (NCT05710198).

Citicoline has become a potential neuroprotective treatment for glaucoma. Although initial preclinical and early clinical studies have shown promising outcomes, larger and more thorough trials are necessary to validate its effectiveness and define its place in clinical practice. The current research will offer important insights into the viability of citicoline as a standard therapeutic option for glaucoma patients.

3.6. Nicotinamide

Nicotinamide, also known as vitamin B3 or NAM, has a simple chemical structure consisting of a pyridine ring with an amide group at the 3-position. This structure allows it to serve as a precursor to nicotinamide adenine dinucleotide (NAD), a vital coenzyme in cellular metabolic processes. NAD is essential for mitochondrial function, as it supports oxidative phosphorylation and ATP production, which are critical for maintaining neuronal health and energy homeostasis. As cellular concentrations of NAD decline with age, mitochondrial dysfunction, inflammation, and oxidative stress worsen, leading to neurodegeneration [113,114,115]. Nicotinamide replenishes NAD levels, supporting mitochondrial function and cellular energy metabolism, while also influencing calcium homeostasis, vascular regulation, and neuronal health, making it a promising candidate for neuroprotection in glaucoma [116,117,118].

A study revealed that the reduced retinal NAD levels caused by aging in mice could be mitigated by high-dose oral nicotinamide [119,120]. Another study showed that oral nicotinamide inhibits RGC loss and nuclear shrinkage [121]. These studies showed that nicotinamide protected RGCs from glaucomatous damage by improving mitochondrial health and reducing oxidative stress. A recent study demonstrated that a combination of NADPH and NAC synergistically reduced apoptosis, axonal damage, and peroxidation in retinal ganglion cells while inhibiting gliosis and p38/MAPK activation in Müller cells, suggesting their potential as a neuroprotective and anti-inflammatory treatment for glaucoma [122]. Novel delivery methods were also investigated and demonstrated a positive result [123]. Similar findings were observed with genetic approaches to overexpress NAD-producing enzymes, such as nicotinamide mononucleotide adenylyl transferase 1 (NMNAT1), NMNAT2, and NMNAT3 [120,124,125]. On the other hand, epigallocatechin gallate (EGCG), a polyphenol found in green tea, promotes NAD production through a mechanism dependent on NMN and NMNAT2, making it a useful compound for developing new drugs [125].

Human studies have also supported the potential of nicotinamide in glaucoma treatment. A study reported significantly reduced plasma NAD levels in patients with primary POAG compared to controls, suggesting a systemic NAD deficiency in glaucoma [126]. This finding aligns with preclinical data and underscores the need for therapeutic strategies to restore NAD levels. A randomized crossover clinical trial (ACTRN12617000809336) showed that oral nicotinamide supplementation enhanced RGC function, as assessed by ERG, regardless of IOP levels [127]. Additionally, a Phase II randomized clinical trial (NCT03797469) found short-term improvements in visual function in patients with moderate OAG who received a combination of nicotinamide and pyruvate [128].

Ongoing clinical trials are exploring the long-term effects of nicotinamide supplementation in glaucoma patients. The Glaucoma Nicotinamide Trial (TGNT) is investigating the neuroprotective benefits of nicotinamide in 660 OAG patients, with an anticipated completion date by the end of 2026 (NCT05275738). Additionally, a Phase III RCT is evaluating the effect of oral nicotinamide on visual field progression over 27 months, with results expected by 2026 (NCT05405868). Another study is examining the neuroprotective potential of nicotinamide riboside, a more bioavailable precursor, over 24 months in 125 POAG patients [129,130]. As research progresses, nicotinamide could become an integral part of glaucoma management, offering hope for improved patient outcomes.

3.7. Insulin

The mTOR pathway is crucial for RGC energy metabolism, with insulin serving as a primary activator of both mTORC1 and mTORC2 [131]. Insulin crosses the blood-brain/retinal barrier and impacts neuronal survival, neurotransmission, and glucose uptake. Impaired insulin signaling is associated with neurodegenerative diseases, including glaucoma [132]. The research indicates that insulin enhances glucose transport, promotes dendritic regeneration, and supports neuronal survival. The activation of mTORC1/2 is vital for insulin-mediated dendrite repair and synapse restoration in RGCs, positioning insulin as a promising therapeutic target [133].

Intranasal insulin has emerged as a promising method for bypassing the blood–brain barrier without causing systemic side effects like hypoglycemia [134]. Research on intranasal insulin in Alzheimer’s and mild cognitive impairment shows that it is a safe and effective way to target insulin signaling in neurodegeneration [135]. Although clinical trials in glaucoma are yet to be conducted, preclinical evidence suggests that exogenous insulin may help preserve RGCs [132]. A Phase 1 trial investigating topical insulin eye drops for glaucoma was conducted and showed safety in patients with glaucoma [136]. Overall, intranasal and topical insulin presents a promising treatment strategy for neurodegenerative diseases, though further research is required to validate its efficacy.

3.8. Resveratrol

Resveratrol (RES), a polyphenolic compound with antioxidant properties, has shown potential in slowing glaucoma progression by supporting RGC health [137]. RES stimulates cell growth, reduces apoptosis, and alleviates oxidative stress, especially in RGCs exposed to hydrogen peroxide [138].

Additionally, RES protects RGC axons by inhibiting JNK protein phosphorylation through Sirt1 activation and prevents oxidative stress-related damage by suppressing the MAPK pathway [139]. RES preserves retinal function by modulating hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), and the p38/p53 pathways, while concurrently activating the PI3K/Akt pathway to promote cell survival [140]. These mechanisms demonstrate RES’s therapeutic potential in protecting RGCs from degeneration and managing glaucoma progression [141]. Intravitreal administration of RES successfully protected RGCs from high IOP-induced cell death, though further research is needed to fully evaluate its efficacy [142].

3.9. Other Novel Drugs

Recent advancements in neuroprotective therapies for glaucoma have highlighted several promising compounds and mechanisms.

Dopamine (DA), produced by dopaminergic amacrine cells (DACs), plays key roles in light adaptation and circadian rhythms [143]. Increasing DA release from DACs, along with overexpressing the Drd1 receptor in all RGCs, significantly improves RGC survival, encourages axon regeneration after optic nerve damage, and protects vision in glaucoma models [144]. Future research using high-throughput single-cell sequencing will be important for understanding the cellular pathways activated by DRD1 [145].

Rho kinase (ROCK) inhibitors, such as ripasudil and fasudil, have shown promising neuroprotective effects in the treatment of glaucoma. These inhibitors not only reduce IOP by enhancing aqueous humor outflow through the trabecular meshwork but also exhibit neuroprotective properties by improving blood flow [146] to the optic nerve and promoting the survival of RGCs [147].

Omidenepag (OMD), an E prostanoid receptor 2 (EP2) agonist, demonstrated neuroprotective effects by suppressing excitotoxic RGC death and modulating glia–neuron interactions via cAMP signaling pathways [148]. The bioprecursor prodrug 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED) selectively generates 17β-estradiol (E2) in the retina following topical application, effectively protecting RGCs and their axons in a male rat glaucoma model without raising systemic E2 levels [149]. The P2X7 receptor antagonist A740003 provided neuroprotection by inhibiting microglial activation, reducing retinal inflammation, and enhancing RGC survival in a chronic intraocular hypertension model [150].

Irisin, an exercise-induced myokine, showed neuroprotective properties by attenuating neuroinflammation and promoting autophagy through integrin αVβ5/AMPK signaling in an acute ocular hypertension model [151]. The novel small molecule H7E, an HDAC8 inhibitor, protected against glaucoma damage by inhibiting Müller glial activation and preventing retinal cell death from oxidative stress [152]. Finally, the Gramine derivative ITH12657 demonstrated significant neuroprotection against excitotoxicity-induced RGC death in a rat model, with specific protection observed in Brn3a+ RGCs and α-ONs-RGCs [153].

Collectively, these studies underscore the potential of diverse neuroprotective strategies in mitigating glaucomatous damage and preserving vision.

4. Emerging Technologies

4.1. Stem Cell Therapy

Stem cell therapy is a promising new approach for treating neurodegenerative diseases like glaucoma. It provides a dual therapeutic benefit by regenerating RGCs and differentiating them into new cell types, while also creating a neurotrophic environment that supports damaged RGCs [154,155].

Mesenchymal stem cells (MSCs), which are multipotent and can differentiate into various cell types such as neurons and glial cells, offer neuroprotective and regenerative effects. They promote neuronal growth, regulate inflammation and immune responses, stimulate angiogenesis, and help reduce demyelination and apoptosis [156,157,158]. MSCs also secrete platelet-derived growth factor (PDGF) and neurotrophic factors (NTFs) like CNTF, FGF-2, GDNF, neuritin, and BDNF, which enhance cell survival and foster the development of other cells [155,159,160,161,162].

Animal models and preclinical studies have shown that MSCs are effective in promoting RGC survival, reducing RGC loss, boosting growth factor expression, enhancing anti-inflammatory properties, and protecting trabecular meshwork tissue when administered intravitreally or intracamerally [160,163,164,165,166,167,168,169]. However, despite these encouraging results in animal models, clinical trials have encountered difficulties. For example, one trial found no significant improvement in visual performance after the intravitreal injection of autologous bone marrow-derived MSCs in a patient with advanced glaucoma. Additionally, another participant in the study developed retinal detachment with proliferative vitreoretinopathy, raising safety concerns [170]. Currently, the Intravitreal Mesenchymal Stem Cell Transplantation in Advanced Glaucoma Study (NCT02330978), a Phase I trial, is assessing the safety of intravitreal MSC injections in advanced glaucoma patients. Other trials, such as the Stem Cell Ophthalmology Treatment Study and its follow-up (NCT01920867, NCT03011541), are examining the variability in patient outcomes based on different MSC delivery techniques.

Human embryonic stem cells (hESCs) are pluripotent and can differentiate into any cell type, making them a potential source for RGCs [171]. Experimental studies have developed protocols for differentiating hESCs into RGCs, which have shown successful integration and the mediation of light responses in preclinical studies involving rat and monkey eyes [172,173,174]. However, the use of hESCs is ethically controversial and scientifically challenging. Other types of stem cells, such as oligodendrocyte precursor cells (OPCs), human neuronal progenitor cells, and retinal stem cells, have also demonstrated potential in protecting RGCs in animal models [175,176]. Mouse-induced pluripotent stem cell (miPSC)/mouse embryonic stem cell (mESC)-derived RGC and spermatogonial stem cell-derived RGC were also investigated [177,178]. Adipose tissue-derived regenerative cells, with their multipotent properties, are being evaluated in clinical trials (NCT02144103) for their safety and efficacy in glaucoma treatment [179].

Despite the potential neuroprotective abilities of stem cells, safety issues remain a significant concern. It is important to carefully evaluate the balance between graft survival and the risk of tumor development, as prolonged stem cell survival raises the likelihood of tumor formation. Moreover, the implanted cells might secrete harmful agents, affecting the microenvironment of RGCs [180]. Adverse effects, including reactive gliosis, vitreous clumping, extensive inflammation, and epiretinal membrane thickening are also concerns [181].

Despite the challenges, stem cell therapy holds significant potential for glaucoma treatment. Ongoing research aims to improve the safety and effectiveness of these treatments. Future investigations should prioritize ensuring the survival and function of transplanted cells, supporting their integration into retinal and brain networks, and maintaining long-term safety. Continued research and larger clinical trials are essential to fully realize the potential of stem cell therapy in glaucoma treatment, paving the way for its integration into clinical practice.

4.2. Gene Therapy

Gene therapy has become a promising strategy for the neuroprotection and treatment of glaucoma. Although currently restricted in clinical use, experimental studies and preclinical research have shown significant potential for gene therapy in managing and potentially reversing glaucomatous damage.

Gene therapy strategies for glaucoma focus on modifying specific genes associated with the disease’s pathogenesis and delivering neuroprotective factors to preserve RGCs. Studies have demonstrated that clustered regularly interspaced short-palindromic repeat (CRISPR)-mediated genome editing of the myocilin (MYOC) gene effectively lowers IOP and inhibits glaucomatous damage in mouse models [182]. Another genetic target under investigation is the tunica interna endothelial cell kinase (TEK), which plays a role in Schlemm’s canal development [183,184]. Additionally, genetic constructs designed to overexpress neuroprotective factors such as BDNF and its receptor TrkB have shown significant neuroprotective effects, enhancing RGC survival and preserving visual function in rodent models. This effect may prove to be a feasible therapeutic strategy in the planned Phase I/II trials [185,186,187,188,189]. In addition to BDNF, other neurotrophic factors such as CNTF have been encoded into genetic constructs to support RGC survival [45,190,191,192].

Other gene targets of different functions have also been investigated [193]. A study reactivating calcium/calmodulin-stimulated protein kinase II (CaMKII) activity in diseased mice resulted in the protection of RGCs and the preservation of visual function [156]. The overexpression of the complement C3 inhibitor shows a neuroprotective effect of RGC axons and somata [194]. The transduction of VEGF variants by VEGFR2 and PI3K signaling promotes synaptogenesis and increases the length of neurites and axons [195]. Combining AAV-γ-synuclein (mScng) promotor with CRISPR/Cas9 gene editing knocks down pro-degenerative genes, preserving the acutely injured RGC somata and axons [196]. The overexpression of Bcl-XL attenuates both RGC soma pathology and axonal degeneration in the optic nerve [197]. The overexpression of NMNAT1 as well as NMNAT2 restores the decreased NAD levels, showing a significant neuroprotective effect of RGC soma, axons, and the maintenance of visual function [120,198]. The overexpression of myc-associated protein X (MAX) prevents RGC death and protects optic nerve axons [199]. The overexpression of the X-linked inhibitor of apoptosis (XIAP) blocks the activation of apoptosis, providing both functional and structural protection of RGCs [200]. The overexpression of SOD2 reduced malonaldehyde, protecting RGCs from oxidative stress [201]. The overexpression of the adaptor molecule Protrudin significantly enhances central nervous system regeneration by promoting the accumulation of essential growth molecules and organelles in distal axons, both in vitro and in vivo. This regeneration is facilitated by Protrudin’s ability to link axonal organelles, motors, and membranes, highlighting its potential as a therapeutic target for axonal injury [202]. NFATc4 knockout in mice enhances RGC survival and delays axonal degeneration following optic nerve injury by suppressing pro-apoptotic signaling, highlighting NFATc4 as a potential therapeutic target for optic neuropathies [203]. Tau overexpression exacerbates retinal degeneration, while Tau silencing offers significant protection, highlighting the critical role of Tau in retinal integrity and its potential as a therapeutic target in glaucoma [204].

Despite promising results in preclinical models, gene therapy for glaucoma faces several challenges. The multifactorial and polygenic nature of glaucoma complicates the identification of effective genetic targets. Additionally, issues related to gene transfer efficiency, site-specific binding, and the potential for mutagenesis need to be addressed. Whole-genome sequencing and advancements in genome editing technology hold the potential to identify new therapeutic targets and refine existing gene therapy approaches. The development of more efficient viral vectors and delivery methods is crucial for translating preclinical successes into clinical practice. With continued advancements in genetic research and therapy, gene therapy may lead to breakthroughs in preserving vision and improving the quality of life for glaucoma patients.

4.3. Mitochondrial-Targeted Therapies and Transplantation

Mitochondria are essential for bioenergetics and play key roles in calcium regulation, cell signaling, apoptosis, and synaptic support. RGCs, with their large dendritic trees and unmyelinated axons in the retina, rely heavily on mitochondria to meet their high metabolic demands. As a result, these cells are highly susceptible to mitochondrial dysfunction, as seen in disorders like Leber hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy [205] In glaucoma models, mitochondrial abnormalities appear before optic nerve degeneration, suggesting that metabolic stress plays a key role in RGC injury and degeneration [206].

Various neuroprotective, mitochondria-targeted therapies for glaucoma have been proposed, including dietary modifications, antioxidant supplementation, stem cell therapy, gene therapy, and mitochondrial transplantation [207]. Citicoline supports mitochondrial function, potentially preventing cell damage, promoting the growth of axons and dendrites, and improving dopaminergic and cholinergic activity [208]. Nicotinamide, through its role in regulating metabolism, has shown significant protection against optic nerve degeneration in animal models of glaucoma [209]. Though clinical evidence for metabolic therapies in glaucoma is limited due to the disease’s slow progression, small studies suggest neuroprotective benefits [128,210].

Mitochondrial transplantation, or mitotherapy, involves transferring functional mitochondria to cells with mitochondrial dysfunction, offering potential treatment for diseases linked to mitochondrial impairment [211]. Studies have shown promising results in animal models, including enhanced oxidative metabolism, neuroprotection, and axon regrowth in RGCs [212]. Human in vitro studies have also demonstrated mitotherapy’s ability to restore cellular function in diseases like LHON [213]. The success of mitochondrial transplantation is influenced by factors like the source and delivery method of the mitochondria, both of which require further investigation.

4.4. Nanotechnologies

Exosomes and nanoparticles are emerging as significant tools in neuroprotective and regenerative strategies for glaucoma treatment. Exosomes, extracellular vesicles released by different cell types, range from 30 to 150 nm in diameter and carry a variety of biologically active substances such as proteins, mRNA, miRNAs, and lipids. They act as intracellular signaling organelles and are involved in numerous pathological processes, including nerve injury and repair, vascular regeneration, and immune response. Their potential therapeutic applications in glaucoma are being increasingly recognized [214,215].

Exosomes derived from MSCs have demonstrated significant neuroprotective effects in glaucoma models. Studies using rat models revealed that bone marrow MSC (BMSC)-derived exosomes significantly increased the survival and axonal regeneration of RGCs through miRNA-dependent mechanisms [166,216]. These exosomes integrated into the inner retinal layers, highlighting the crucial role of miRNAs in their neuroprotective benefits. Additionally, human umbilical cord MSC-derived exosomes have been reported to promote RGC survival and glial activation in rat models [217]. These exosomes also preserved RGCs in human retinal explants post-axotomy by releasing growth factors, especially PDGF [160]. Another recent study demonstrates that small extracellular vesicles (sEVs) derived from MSCs overexpressing microRNA-22-3p (miR22) protect retinal RGCs from apoptosis and preserve retinal function in an NMDA-induced RGC injury model by inhibiting the MAPK signaling pathway. The findings suggest that miR22-sEVs could be a promising therapeutic approach for glaucoma and other diseases involving RGC damage [218]. Despite these promising findings, treatments involving BMSC-derived exosomes and human MSC-derived exosomes have been associated with extensive gliosis and inflammation, necessitating further research to optimize their therapeutic potential and minimize adverse effects [169].

Nanoparticles offer another promising approach for neuroprotection in glaucoma. These tiny particles can encapsulate drugs, siRNA, mRNA, or other nucleic acids, providing a hydrophobic environment that improves the solubility of drugs with low water solubility. Nanoparticles protect encapsulated drugs from hydrolytic degradation and improve their transport across biological barriers, making them effective drug carriers. The use of pharmaceutical nanoparticles also reduces the side effects associated with traditional topical treatments [219].

However, most nano eye drops designed for neuroprotection are still in the animal experimental stage. A study developed magnetic nanoparticles conjugated with neurotrophins. These nanoparticles protect neurotrophins from rapid degradation and facilitate their localization in the retina [220]. Other studies on rodent models suggested that GDNF delivered by nanoparticles could serve as a neuroprotective tool for treating glaucomatous optic neuropathy [221,222]. Another study demonstrates that circular RNA (circRNA)-based therapy using lipid nanoparticle (LNP)-formulated circNGF provides prolonged NGF protein expression and superior protection for RGCs compared to traditional NGF protein therapy, without retinal toxicity [223]. The other study created a topical formulation using tocopheryl polyethylene glycol succinate (TPGS) and Pluronic F127 curcumin nanocarriers, which demonstrated neuroprotective effects in rodent models of ocular hypertension, optic nerve disease, and partial optic nerve transection [224]. Additionally, a study explores the development of polymeric microparticles to sustain the release of hydrogen sulfide (H₂S) for treating glaucoma. The research identifies critical process parameters that affect particle size, entrapment efficiency, and release profile, successfully optimizing a formulation that provides sustained H₂S release for up to 30 days, with potential for both ocular hypotensive and neuroprotective effects [225]. Another study demonstrated that NAM-loaded extracellular vesicles (NAM-EVs) can effectively deliver NAM to RGCs, providing a neuroprotective effect and maintaining the health and function of RGCs [123]. The other study investigated the use of liposomal carriers to enhance the delivery and therapeutic efficacy of citicoline in treating glaucoma, showing promising results for improved bioavailability and sustained drug release in ocular applications [112].

Exosomes and nanoparticles represent innovative and promising strategies for neuroprotection and regeneration in glaucoma treatment. Further human studies are essential to fully elucidate the therapeutic potential of these technologies and optimize their applications in clinical settings, ultimately aiming to improve outcomes for glaucoma patients.

5. Conclusions

Glaucoma continues to pose a major global health challenge, requiring the development of innovative neuroprotective strategies to complement existing IOP-lowering treatments. Pharmacological interventions such as brimonidine, neurotrophic factors, memantine, Ginkgo biloba extract, citicoline, nicotinamide, insulin, and resveratrol have demonstrated promising neuroprotective effects in preclinical studies, with some showing positive results in early clinical trials. Emerging technologies, including stem cell therapy, gene therapy, mitochondrial-targeted therapies, and nanotechnologies, offer innovative approaches to neuroprotection and RGC regeneration. Despite the promising potential of these strategies, further research is required to address the challenges associated with their clinical application. Larger, long-term clinical trials are essential to confirm the efficacy and safety of these treatments, ensuring their successful integration into clinical practice.

The outlook for neuroprotective therapies in glaucoma is promising, but significant hurdles remain in translating preclinical success into routine clinical use. As these novel therapies continue to be explored, it is likely that targeted treatments combining pharmacological agents and cutting-edge technologies could become part of a comprehensive glaucoma management plan. In the future, personalized medicine approaches based on genetic and molecular profiling may further refine these treatments, offering more tailored and effective solutions for preventing vision loss in glaucoma patients.