Glaucoma is classified into primary and secondary forms based on the presence or absence of pre-existent pathological conditions such as uveitis, neoangiogenesis, traumas, and lens abnormalities [2,21]. Additionally, glaucoma can be categorized as either open-angle or angle-closure, based on the chamber angle located between the iris and the posterior surface of the cornea [22]. In a healthy state, this angle is physiologically open, allowing the outflow of aqueous humor (AH) through the trabecular meshwork (TM) to the uvea and conjunctiva, maintaining normal turnover [23,24]. Primary open-angle glaucoma (POAG) is the most common form of glaucoma [7], and is often associated with high IOP. However, it also includes a subtype known as normal-tension glaucoma (NTG), in which the IOP is not elevated. NTG accounts for 30–90% of POAG cases and its prevalence varies significantly depending on geographical location [11,25]. Possible explanations for this significant difference have been attributed to an alternative risk-factor profile found in different populations, such as genetic components [26], long axial length [27], low intracranial pressure, and vascular dysregulation [25].

In the context of primary angle-closure glaucoma (PACG), there is anatomical contact between the iris and the cornea, and in 90% of cases, between the iris and the lens, creating a pupillary block [28]. Although PACG cases account for approximately 26% of total glaucoma cases [29], they are responsible for approximatively half of worldwide cases of glaucoma-related blindness [6,30].

From an epidemiological standpoint, it was estimated that in 2013, approximately 64.3 million people between the ages of 40 and 80 were affected by glaucoma. However, by the year 2040, it is projected that the number of individuals affected will exceed 110 million [7]. The direct costs associated with this condition are primarily linked to disease progression and the need for treatment adjustments when initial therapies are unsuccessful, contributing to cost escalation [31]. Indirect costs, such as the loss of well-being and visual disability experienced by patients, have been estimated to be the most impactful economic factors in Europe [32]. Additionally, glaucoma has been shown as a possible risk factor for falls which require hospitalization [33]. Collectively, numerous studies on this subject have emphasized the importance of halting disease progression and preventing late-stage glaucoma to minimize the loss of well-being for patients and to prevent escalating costs.

Glaucomas are chronic and progressive optic neuropathies that, if left untreated, can potentially lead to irreversible visual loss. According to the existing literature, 15 to 20% of patients with glaucoma may experience unilateral blindness [34,35,36,37]. The prognosis can vary depending on the subtype of glaucoma [6,38]. However, it is important to note that the majority of patients with glaucoma can maintain useful vision through appropriate treatments aimed at lowering IOP [39,40]. The early detection and management of glaucoma, along with regular follow-ups and adherence to treatment regimens, play a crucial role in preserving vision and delaying disease progression.

POAG and PACG present with different sets of symptoms. In POAG, the disease progression is often asymptomatic due to binocular compensation. As a result, patients typically experience the first noticeable symptoms only in advanced stages when significant damage to the visual field has already occurred [22,41].

On the other hand, PACG manifests with rapid and painful symptoms. Affected individuals may experience a rock-hard sensation in the eye, corneal edema, reduced visual acuity, conjunctival hyperemia (redness), irradiating pain, and potentially accompanying nausea and vomiting [22]. PACG is considered an ophthalmologic emergency that necessitates immediate medical intervention to prevent severe visual loss [22].

The use of appropriate diagnostic tools is crucial for facilitating the detection of early signs of glaucoma and initiating prompt and appropriate therapy to prevent further damage. Tonometry, fundoscopy, and perimetry are valuable in enabling an early diagnosis [22,42,43]. Classic fundoscopic signs of glaucoma include an enlarged optic cup, resulting in an increased cup–disc ratio, the loss of the neuroretinal rim, the presence of disc hemorrhages, and parapapillary tissue atrophy [22,43].

Assessing the disease progression of glaucoma can be achieved through an examination of the neuroretinal rim of the optic nerve head using fundoscopy or through perimetric evaluation [44]. Recently, SD-OCT has also been described as a suitable diagnostic tool for staging glaucoma [45]. However, despite the availability of various diagnostic features for assessing glaucomatous disease progression, there is currently no consensus on a singular criterion to determine the specific disease stages [46].

The primary objective of the major established antiglaucoma drugs is to reduce IOP to a personalized and acceptable range to halt the progression of the disorder [47]. These medications are typically administered topically via eye drops, and can be categorized based on their pharmacological mechanisms into the following groups:

• Prostaglandin analogues: examples include bimatoprost, which enhances both trabecular and uveoscleral outflow of AH [22].
• β-blockers: medications like levobunolol and timolol work by reducing the production of AH [22].
• α₂-adrenoceptor agonists: drugs such as apraclonidine and brimonidine lower IOP by decreasing aqueous humor production and augmenting trabecular outflow [22].
• Carbonic anhydrase inhibitors: agents like brinzolamide act by reducing the production of aqueous humor [22].
• Miotic agents: pilocarpine, for instance, increases the chamber angle by constricting the pupil and can also provide neuroprotective effects through the activation of muscarinic receptors [48].
• Rho-associated protein kinase (ROCK) inhibitors: Netarsudil is a ROCK inhibitor that targets the ROCK pathway, suppressing fibrotic events in the trabecular meshwork ™ and optimizing aqueous humor flow, thereby reducing IOP [49]. This molecule has been approved for the treatment of glaucoma in the United States (2017) and Europe (2019) in the form of a 0.02% ophthalmic topical formulation for once-daily application [50].

Additionally, laser and surgical procedures are well-established in clinical practice for the management of both open-angle and angle-closure glaucoma. These therapeutic options have the goal of improving the outflow of AH or to reduce its production [4,22,51,52]. Surgical interventions in glaucoma are usually considered a second-line therapy when conservative options fail to sufficiently lower IOP. These surgical options are, for example, cyclocryocoagulation, minimally invasive procedures such as stent implantation, and filtering procedures like trabeculectomy [22]. Due to the scarring processes that may affect the long-term efficacy of surgical techniques bypassing the outflow of AH to subconjunctival spaces, medications are often employed postoperatively to inhibit excessive scar tissue growth [53]. Commonly used medications for this purpose in clinical practice include topical steroids and non-steroidal anti-inflammatory drugs. Off-label drugs, such as 5-fluorouracil and mitomycin C, are also utilized [53]. Additionally, there are ongoing investigations into the use of biologic drugs, such as bevacizumab (anti-vascular endothelial growth factor, VEGF), as well as molecules targeting the transforming growth factor (TGF-β) signaling pathway, like lerdelimumab (anti-TGF-β2) and decorin (a proteoglycan that also targets TGF-β signaling) [53].

The main risk factor for both POAG and PACG is an elevated IOP, defined as a pressure value above the 97.5th percentile for the specific population under consideration, often considered to be higher than 21 mmHg [2,54]. In addition to IOP, other risk factors for POAG include myopia, advanced age, belonging to the black ethnic group, and a family history of the condition. For PACG, risk factors include being female, having a small corneal diameter, hyperopia, an anteriorly positioned lens, and shallower central and limbal anterior chamber depth [29]. However, IOP is recognized as the primary modifiable risk factor, making it the main target of current established antiglaucoma drugs [22].

Two major theories have been proposed to explain the pathogenesis of glaucoma, both emphasizing the association between an elevated IOP and the development of the disease: the “vascular” and the “mechanical” theories. According to the vascular theory, a high IOP leads to compression of the blood vessels supplying the ONH, resulting in reduced blood flow, hypoperfusion, and subsequent ischemia in RGCs [55,56]. On the other hand, the mechanical theory suggests that an elevated IOP causes compressions and deformations of the lamina cribrosa and RGC axons, initiating a cascade of events that lead to cell death due to blocked axoplasmic traffic and inadequate cellular supply [57]. Figure 1 provides a summary of the events leading to mechanical damage in RGCs as the consequence of an elevated IOP.

An elevated IOP is proposed to arise from a pathological increase in resistance to AH flow within the TM [58]. The TM, located in the chamber angle, consists of three layers: the uveal TM, corneoscleral TM, and the juxtacanalicular TM (also known as the cribriform TM region), which borders Schlemm’s canal. AH flows through the TM and reaches the episcleral veins of the conjunctiva via the Schlemm’s canal [59]. The permeability of the TM to AH plays a crucial role in regulating IOP levels [59]. Structural alterations in the TM can lead to the apoptosis of TM cells and the disintegration of its structure [60]. Additionally, changes in the deposition of the extracellular matrix within the TM can disrupt the adhesion of TM-endothelial cells [61]. TGF-β2 appears to play a pivotal role in promoting the deposition of the extracellular matrix within the human TM during glaucoma [62]. These events ultimately result in increased resistance to AH drainage within the TM, leading to an elevated IOP [60].

In the “Collaborative Normal-Tension Glaucoma Study”, a clinical trial, the effectiveness of IOP-lowering therapy in NTG was evaluated. The study revealed that although reducing IOP can have a positive impact, it alone cannot completely halt disease progression [12,63]. This suggests the involvement of additional factors in the development of NTG. The wide geographical variability in the prevalence of NTG and the relatively high percentage (approximately 21%) of patients reporting a family history of the condition [64] suggest a possible genetic predisposition. Individuals with NTG may have a lower tolerance for what are considered “normal” IOP levels [12]. The increased susceptibility of RGCs to IOP-induced damage is believed to contribute to the mechanical injuries observed in NTG, similar to those seen in glaucomas associated with an elevated IOP [12]. Numerous specific gene polymorphisms, resulting in the altered functionality of corresponding proteins, have been associated with NTG [64]. For example, certain sequence variants of the optineurin (OPTN) gene, which encodes a neuroprotective and IOP-regulating protein, have been linked to NTG [65]. Zhu and colleagues have provided evidence supporting the neuroprotective role of OPTN in RGCs by counteracting inflammation and apoptosis. They found that OPTN negatively regulates the tumor necrosis factor-α (TNF-α)-induced NF-κB activation, which plays a crucial role in cell survival [66]. Additionally, Minegishi et al. extensively reviewed the significance of OPTN in glaucoma [67]. Specifically, they focused on the most common OPTN mutation in NTG, known as E50K, and highlighted its impact in triggering abnormal aggregation of intracellular vesicles [68,69]. Moreover, the E50K mutation was associated with a disruption of the Golgi structure, leading to cellular toxicity [70,71,72,73]. In addition, mutations in the optic atrophy type 1 (OPA1) gene, which is crucial for mitochondrial dynamics, have also been implicated in the pathogenesis of NTG. These mutations can lead to RGC apoptosis through mitochondrial dysfunction [64,74]. Furthermore, specific gene sequence variants of the endothelin-1 (ET-1) receptor A have been identified as being associated with NTG [75].

In addition to genetic factors, several alternative risk factors have been identified as potential contributors to the pathophysiology of NTG, including systemic vascular dysregulation, oxidative stress, and endothelial dysfunction [11,12,17]. A systemic vascular impairment, such as cerebral silent infarcts and nocturnal arterial hypotension, has been associated with NTG, potentially leading to the condition of hypoperfusion in the ONH [11,12,76,77,78]. Hypoperfusion-induced hypoxia may initiate the glaucomatous pathogenesis of NTG [79]. As a result of hypoxic insults, the hypoxia-inducible factor 1α (HIF-1α), a potent cytokine, triggers downstream transductions that activate glial cells, leading to neuroinflammation, similar to the events observed in glaucoma associated with a high IOP [14,80,81]. Vascular endothelial dysfunction is another characteristic of NTG and may manifest through the impairment of vasoregulatory factors, such as nitric oxide (NO) [82] and ET-1 [64]. Excessive reactive oxygen species (ROS) can reduce NO-dependent vasorelaxation due to the impaired activity of endothelial nitric oxide synthase (eNOS). In the context of an altered redox status, the fundamental cofactor of eNOS, tetrahydrobiopterin, undergoes oxidation to dihydrobiopterin, resulting in abnormal eNOS activity, the production of peroxynitrite (ONOO⁻), and a lower bioavailability of NO [82]. Consequently, dysfunctional vasoregulation occurs, leading to deficits in vasorelaxation [64,83,84]. Moreover, the vasoconstrictor peptide ET-1 has been reported to be increased in the plasma [85,86] and in the AH [87] of NTG patients. The abnormal vasoconstriction induced by ET-1 may affect the blood vessels supplying the ONH in NTG, further contributing to reduced perfusion [88]. The combined processes of decreased NO-dependent vasodilation and increased ET-1-induced vasoconstriction in blood vessels may result in a reduced perfusion of the ONH, forming the etiopathogenic basis for primary damage to RGCs in NTG [64,89]. Figure 2 illustrates the pathomechanisms due to increased susceptibility to IOP in RGCs, to hypoperfusion, and to endothelial dysfunction in NTG.

Hypoxia, which occurs during low oxygen concentrations, can also contribute to ROS production as the electron transport chain slows down, leading to an accumulation of reducing equivalents and subsequent O₂^•− production [92]. Hypoxia-inducible factor 1-alpha (HIF-1α) is upregulated in response to hypoxia in glaucoma patients and can further enhance NOX-2 and inducible nitric oxide synthase (iNOS) expression, resulting in ROS production [13,93,94,95,96]. ROS, in turn, can trigger the expression of HIF-1α, creating a feedback loop that amplifies inflammation and apoptosis [97,98]. Glial activation and the release of TNF-α follow, leading to the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), a transcription factor responsible for inflammation. This process amplifies glial activation, neuroinflammation, and ultimately apoptosis [14,80,81]. The activation of the apoptosis signal-regulating kinase 1 (ASK-1)/p38 mitogen-activated protein kinase (MAPK)/JNK/extracellular-signal-regulated kinase (ERK) axis by ROS can lead to caspase-3 activation and cellular membrane disassembly, promoting cell death [99,100]. Chronic exposure to ROS can activate the phosphoinositide 3-kinase (PI3K)/Akt axis while attenuating the mammalian target of rapamycin (mTOR) pathway, further stimulating the NF-kB and enhancing inflammatory events [101]. Additionally, excessive ROS disrupts glutamate metabolism, leading to the neurotoxic extracellular accumulation of glutamate, as dysfunctional glial cells fail to properly buffer the excess glutamate [16,102,103]. Moreover, oxidized metabolites, like advanced glycation end products (AGEs) and oxidized low-density lipoproteins (oxLDLs), can act as “antigenic” stimuli, promoting ROS production, NF-kB activity, glial activation, and apoptosis [15,16].

Mitochondrial dysfunction plays a central pathophysiological role in glaucoma and is associated with inflammation, oxidative stress, impaired mitochondrial dynamics and reduced ATP production [104]. Excessive ROS and metabolic stress due to a nutrient deficit can lead to mtDNA mutations and subsequent mitochondrial dysfunction [105]. Mechanical insults from an elevated IOP can cause mitochondrial disruptions and deficiencies in the OPA1 gene, which regulates mitochondrial fusion, a process important for mitochondrial quality control [15,104,106,107,108]. A deficiency in OPA1 can trigger ROS overabundance and glutamate excitotoxicity [109]. Conversely, the upregulation of OPA1 has been shown to have a protective effect on RGCs by enhancing mitochondrial fusion and mitophagy, the selective autophagy of damaged mitochondria [110,111,112]. In glaucoma, the balance between mitochondrial fusion and fission is disrupted, leading to increased fission and reduced fusion and mitophagy, which results in an elevated mitochondrial number and decreased mitochondrial size [113,114,115]. In humans, mitochondrial fission is mainly mediated by the dynamin-related protein 1 (Drp-1) [116]. In a murine model of glaucoma, an elevated IOP leads to the dephosphorylation of Drp-1, resulting in mitochondrial fragmentation and RGC loss via apoptosis [117]. A recent in vitro study demonstrated that an ERK1/2-Drp1-ROS axis induced by an elevated IOP could trigger mitochondrial dysfunction and apoptosis in RGCs [118]. Furthermore, oxidized mitochondrial DNA and mitochondrial fragments released from microglia can activate the NLRP3 inflammasome, leading to the production of inflammatory cytokines [15,106,119]. Mitochondria are also involved in glial neuroinflammation processes through the mitochondrial ROS-generated activation of NF-κB, leading to the production of inflammatory cytokines [15].

The endoplasmic reticulum (ER) and mitochondria interact through calcium-dependent processes, influencing each other and leading to energy deficiency, apoptosis, inflammation, and increased ROS production [102]. The ER is an intracellular organelle responsible for protein processing and folding to ensure their proper functionality [120,121]. Various conditions such as oxidative stress, protein mutations, viral infections, nutritional deficits, and hypoxia can impact the ER, leading to an accumulation of unfolded proteins [122,123,124]. This results in ER stress, triggering the unfolded protein response (UPR) to restore cellular homeostasis [125]. Chronic ER stress can paradoxically perpetuate UPR activation, leading to apoptosis, the activation of NF-kB, and further ROS formation [123,124]. The UPR consists of three main signaling pathways:

• The inositol-requiring protein 1 (IRE-1)/spliced X-box binding protein-1 (sXBP1)/Janus Kinase (JNK) pathway, which improves protein folding but can also induce inflammation and apoptosis [120,124,126].
• The protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)/activating transcription factor 4 (ATF4)/CCAAT-enhancer-binding protein homologous protein (CHOP) pathway, which reduces protein translation but can increase ROS production and promote apoptosis [127].
• The activating transcription factor 6 (ATF-6) pathway, which enhances the elimination of misfolded proteins and optimizes protein folding but may also activate proapoptotic cascades [120,124,128].

The ROS generated during ER stress, particularly through the ATF4/CHOP pathway, can activate inflammasomes, leading to increased neuroinflammation and further damaging mitochondria [127].

Elevated hydrostatic pressure and ischemia can trigger the release of the major proinflammatory cytokine, TNF-α, from the glial cells, initiating inflammation and apoptosis in RGCs [80]. TNF-α plays a pivotal role in glaucomatous inflammation and oxidative processes. It is secreted by microglia, astrocytes, and Müller cells and contributes to mitochondrial dysfunction, increased ROS levels, and NF-kB expression, which in turn promote the expression of proinflammatory cytokines and adhesion molecules [14,80]. Heat shock proteins (HSPs) and mitochondrial damage-associated molecular patterns (DAMPs) have been investigated as “highly antigenic molecules” associated with neuroinflammation in glaucoma [129,130,131,132]. These molecules can activate the Toll-like receptors (TLRs) expressed in glial cells, leading to NF-kB activation and neuroinflammation [72,130]. The dysregulation of the complement system and the infiltration of activated T cells and monocytes have also been implicated in RGC death [129,133,134,135,136]. Indeed, studies conducted on murine glaucoma models have provided evidence that the absence of the complement can attenuate disease progression [137,138]. In recent years, a novel process called necroptosis has been introduced as the mechanism responsible for axonal degeneration in neurodegenerative disorders. This process can be triggered by TLR-, Fas-, TNF-α-, and interferons (IFNs)-signaling, and is characterized by cell swelling, granular cytoplasm, and cellular lysis [139]. Unlike apoptosis, which typically involves caspases, necroptosis relies on kinase-mediated transductions [140]. Importantly, apoptotic cell death is generally immunosuppressive, while necroptotic cell death triggers inflammation [140]. Ko and co-workers recently demonstrated in an experimental neuroinflammatory model of glaucoma that TNF-α can exclusively induce necroptosis in axons. This process is dependent on the presence of sterile alpha and TIR motif 1 (SARM1), and involves the reduction of axon survival factors, such as nicotinamide mononucleotide adenylyltransferase 2 and stathmin 2. Additionally, the activation of SARM1 NADase leads to calcium influx and subsequent axon degeneration [141].

Collectively, new insights into the neuroinflammatory processes highlight the role of microbiota via TLR-signaling and of specific programmed cell death pathways, like SARM1-dependent necroptosis, which require a more complete understanding to possibly transfer this new knowledge into the design of experimental immunomodulatory strategies.

Evidence of autoimmune factors in glaucoma has been described, with autoantibodies detected in the sera and retina of glaucoma patients [17,142,143,144,145,146,147,148,149,150,151]. Heat shock proteins (HSP) may play a critical role in this context. HSPs can be produced by bacteria or generated endogenously by cells at the sites of inflammation, and they can activate specific HSP-induced T-regulatory cells [152]. High levels of HSP autoantibodies, including antibodies against HSP27, have been found in the sera of glaucoma patients. These autoantibodies have been shown to trigger neuronal apoptosis by interfering with the function of native HSPs in stabilizing the cytoskeleton [153,154,155]. Autoantibodies against HSP60 [156] and HSP70 [157] have also been detected in the sera of glaucoma patients [158]. Furthermore, studies have demonstrated IgG autoantibody depositions in glaucomatous retinas, along with an increase in CD27+/IgG+ plasma cells and elevated levels of TNF-α, IL-6, and IL-8. These proinflammatory mediators were found to be released by activated microglia [142].

On the other hand, glaucoma patients have shown the downregulation of protective, naturally occurring autoantibodies against 14-3-3 and γ-synuclein, which may contribute to secondary injuries in RGCs [158,159].

Taken together, and considering the sequence of the pathogenetic events, imbalances between pro-apoptotic and anti-apoptotic autoantibodies in autoimmune responses may contribute to secondary injuries in RGCs [158,160]. The autoantibodies found in glaucoma patients may serve as useful diagnostic biomarkers [161].

Figure 3 summarizes the processes leading to the loss of RGCs in glaucoma.