Transforming growth factor beta (TGF-β) is a protein belonging to a larger family called the transforming growth factor beta superfamily, which includes bone morphogenetic proteins (BMPs), inhibins, activins, differentiation growth factors and other signaling molecules (Shi and Massagué, 2003; Massagué, 2012). TGF-β is a cytokine involved in signaling cascades related to cell differentiation, cell division, chemotaxis, and fibrosis. TGF-β has three isoforms: TGF-β1, TGF-β2, TGF-β3, of which TGF-β2 is most associated with eye function (De Caestecker, 2004). The cellular effect of TGF-β signaling depends on the type of protein isoform and its tissue localization. One of the effects of TGF-β is increased extracellular matrix production and remodeling.

The canonical TGF-β signaling pathway begins with its binding to the type II receptor, which leads to phosphorylation and activation of the type I receptor. This process activates intracellular SMADs proteins (Smad2, Smad3, Smad4), which form an oligomeric complex with co-SMAD (Feng and Derynck, 2005; Massagué et al., 2005). The Smad complex moves to the cell nucleus, where it activates gene transcription, leading to the production of extracellular matrix. In the healthy eye, TGF-β2 is involved in corneal healing and scar formation (De Caestecker, 2004). Studies in mice have shown that overexpression of TGF-β2 induces an IOP elevation in wild-type mice, while this effect was not observed in Smad3 knockout mice – thus inferring the importance of Smad proteins in the molecular mechanisms of glaucoma development (McDowell et al., 2013; Figure 2).

Studies have shown that another important regulator, involved in TGF-β2-induced IOP increase, is Src kinase. The application of a potent Src inhibitor, Dasatinib, led to the mitigation of TGF-β2-induced IOP elevation in rat eyes (Tsukamoto et al., 2019). Moreover, investigations conducted on human trabecular meshwork cells demonstrated that TGF-β2 activates Src signaling, promoting enhanced cell adhesion, cytoskeletal remodeling, and the accumulation of extracellular matrix (ECM). Furthermore, the use of a Src inhibitor attenuated TGF-β2-induced changes. Src activity plays an important role in the TGF-β-induced elevation of intraocular pressure (IOP). Identifying Src signaling as a potential therapeutic target in glaucoma suggests promising avenues for intervention.

Neurotrophins (NTs) are central regulators, exerting a critical influence on diverse signaling pathways essential for both neuronal survival and differentiation. Additionally, they play a significant role in promoting synaptic plasticity and facilitating axon regeneration (Rudzinski et al., 2004; Skaper, 2008; Mallone et al., 2020). The neurotrophic pathways are also involved in the development of glaucoma (Rudzinski et al., 2004; Johnson et al., 2011; Weissmiller and Wu, 2012). The neurotrophic pathways provide support and protection to RGCs, and the loss of this support can contribute to the death of RGCs in glaucoma (Guo et al., 2014; Nuschke et al., 2015; Oddone et al., 2017; Dias et al., 2022; Figure 2). Recent literature highlights that heightened intraocular pressure (IOP) and oxidative stress are implicated in the reduction of available neurotrophic factors, ultimately contributing to the demise of retinal ganglion cells (RGCs) in glaucoma. The neurotrophin family comprises pivotal members such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4/5 (NT4/5). Neurotrophin activities primarily hinge on the activation of two distinct transmembrane glycoproteins: tropomyosin receptor kinases (TrkA, TrkB, and TrkC) and the low-affinity neurotrophin receptor p75NTR. Trk activation triggers intricate signaling cascades, activating the Ras/ERK (extracellular signal-regulated kinase) pathway and stimulating mitogen-activated protein kinases (MAPs). Simultaneously, the PI-3 kinase/Akt pathway engages, and phospholipase C (PLC)-γ1 is activated. The activation of p75NTR arises from the binding of immature precursor forms of neurotrophins (proNT), eliciting a dual effect on cell fate-either apoptosis or survival. This outcome is contingent upon the simultaneous expression of Trk receptors. Consequently, maintaining cellular homeostasis hinges on achieving equilibrium in the availability of proNTs and their mature counterparts, alongside the presence of both Trk and p75NTR receptors (Levi-Montalcini et al., 1996; Frade and Barde, 1998; Skaper, 2008; Al-Qudah and Al-Dwairi, 2016; Platholi and Lee, 2018).

Nerve growth factor (NGF), the inaugural and extensively studied member of the neurotrophins (NTs) family, plays a pivotal role in neuronal differentiation and survival. Numerous studies highlight the influence of NGF on these processes, particularly within the optic nerve and visual pathway. Expression of NGF is observed in key ocular structures such as the retinal pigment epithelium, Müller cells, and retinal ganglion cells. Correspondingly, NGF receptors are expressed in these structures as well as in photoreceptors (Chakrabarti et al., 1990). Experimental investigations underscore the significance of NGF in promoting the survival and development of RGCs through its role as a local paracrine and autocrine mediator (Colafrancesco et al., 2011; Allen et al., 2013; Garcia et al., 2014; Roberti et al., 2014; Wang et al., 2014; Aloe et al., 2015; Chen Q. et al., 2015; Mallone et al., 2020; Li et al., 2022; Lin et al., 2022). NGF is delivered retrogradely along the axons of RGCs (Dias et al., 2022). Sposato et al. (2009) treated rats having elevated IOP with topical application of NGF. The results showed that increased IOP alters the basal content of NGF and NGF receptors in visual centers in brain and that NGF topical application normalized these deficits. These findings suggest that degeneration in retinal areas may contribute to a reduction in NGF levels. Following above, the clinical trial of topical application of NGF in glaucoma has been initiated (Beykin et al., 2022).

Another of NTs is brain-derived neurotrophic factor (BDNF), which, due to its potential neuroprotective effects, is the subject of much research in developing therapies for diseases such as Parkinson’s disease, amyotrophic lateral sclerosis, Alzheimer’s disease, and glaucoma (Arancibia et al., 2008; Greenberg et al., 2009; Allen et al., 2013; Domenici et al., 2014; Kimura et al., 2016). Synthesized in the brain, BDNF undergoes retrograde transport to RGCs (Dias et al., 2022). Impaired axonal transport from the brain to RGCs is considered a critical factor contributing to RGC death and optic nerve degeneration in glaucoma. BDNF is also locally produced in the ganglion cell layer (GCL), the inner nuclear layer, and in Müller cells. Notably, retinal BDNF levels were observed to increase in a pressure-induced animal model of glaucoma (Guo et al., 2010). Increased retinal BDNF level was shown to be neuroprotective in optic nerve injury models, including glaucoma (Ritch and Sharma, 2000; Arancibia et al., 2008; Domenici et al., 2014; Sanna et al., 2017; Wójcik-Gryciuk et al., 2020). BDNF plays a crucial role in maintaining the integrity of the inner retina under normal conditions, and its deficiency has been linked to retinal and optic nerve damage during progressive glaucoma (Gupta et al., 2014). The functional activity of BDNF is manifested through its interaction with the TrkB receptor, leading to the activation of intracellular signaling pathways associated with cell viability, including the phosphatidylinositol-3 kinase (PI-3 K)/Akt and extracellular signal-regulated kinases 1/2 (Erk1/2) pathways. Both of these signaling pathways have been implicated in experimental glaucoma (Cheng et al., 2002; Carlotta et al., 2016; Liang et al., 2019). There are several studies using different models of glaucoma that indicate that local administration of BDNF to the eye results in reduced loss of RGCs. Moreover, there is speculation that combining BDNF administration to the eyeball with administration to visual centers in the brain could enhance the survival of RGCs in animals with induced optic nerve damage (Weber et al., 2010; Weber and Harman, 2013).

Several studies suggest that glaucoma development has analogies with neurodegenerative diseases connected with inflammatory responses (Yang et al., 2011; Olmos and Lladó, 2014; Wilson et al., 2015; Parsadaniantz et al., 2020; Rolle et al., 2021; Wong and Benowitz, 2022). In this chapter, we will discuss the possible molecular mechanisms of neuroinflammation development (Figure 2).

Within the retina, microglia and astroglia emerge as pivotal players in the orchestration of inflammatory processes (García-Bermúdez et al., 2021; Quan et al., 2022). Their function is to regulate ion concentration, metabolic support of neurons and neuroprotective activity. Microglia cells, once mature, can be activated by damage-associated molecular pathways (DAMPs), which are released by neuronal cells (Gülke et al., 2018). In addition, increased IOP stimulates RGCs to produce and secrete heat shock proteins (HSPs; Piri et al., 2016). These factors stimulate microglial cells to secrete cytokines and chemokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and complement factors, which promote the morphological transformation of microglia into M1 (proinflammatory) and M2 (anti-inflammatory) macrophages (Li and Barres, 2018). Microglial cells communicate with astroglia by secreting signaling molecules that can lead to the differentiation of astroglia cells into two phenotypes: harmful A1 and neuroprotective A2 (Li et al., 2020). Both of these cell types, microglia and astroglia, are significantly involved in the formation and regulation of neuroinflammation.

Inflammatory pathways are involved in the regulation of various genes that are upregulated in the retina. One of the regulatory factors is nuclear factor-kappa B (NF-κB) – it leads to upregulation of IL-1 cytokine family, IL-6 and TNF-α. TNF-α and another proapoptotic protein, FasL, have been implicated in the pathogenesis of glaucoma (Gregory et al., 2011). Recent research has focused on identifying new therapeutic targets that can reduce or completely abolish the deleterious effects of neuroinflammation.

Astroglial NF-κB has been identified as one of the possible therapeutic targets for modulating the inflammatory response (Yang et al., 2020). The experimental use of mice with induced glaucoma and with or without deletion of astroglial IκKβ, which is the main activating kinase involved in IκB degradation through the canonical pathway of NF-κB activation, showed reduced production of inflammatory cytokines in brain ganglia lacking IκKβ, including TNF-α, which can induce apoptosis of RGCs and axonal degeneration in glaucoma.

Another target of research is Fas/FasL signaling pathway. Fas ligand (FasL) is a protein that belongs to the TNF family. When bound to the Fas receptor, it causes induction of apoptotic and pro-inflammatory pathways (Ju et al., 1995; Gregory et al., 2011). This pathway has been identified as involved in axonal degeneration, death of RGCs and induction of neuroinflammation in a chronic and induced mouse model of glaucoma (Krishnan et al., 2016, 2019) Another study used the small peptide molecule ONL1204, an antagonist of the Fas receptor, which was administered to mice by injection into the vitreous body (Krishnan et al., 2019). The results showed reduced death ratio of RGCs in high-tension glaucoma model. Reduced expression of genes involved in the pathogenesis of glaucoma was also observed, including chemokines and cytokines (GFAP, Caspase-8, TNFα, IL-1β, IL-6, IL-18, MIP-1α, MIP-1β, MIP-2, MCPI, and IP10), components of the complement cascade (C3, C1Q), Toll-like receptor pathway (TLR4).

Recent studies in animal models suggest that monocyte-like cells enter the optic nerve head in an ocular hypertensive mouse model of glaucoma (DBA/2 J) and produce damaging molecules that lead to neurodegeneration (Howell et al., 2012; Wilson et al., 2015; Williams et al., 2017). However, the mechanisms of this action are not fully understood. To study these interactions, monocyte infiltration inhibitors were used: DS-SILY, a peptidoglycan that provides a barrier to platelet adhesion to the vessel endothelium and to monocytes, and genetic targeting of Itgam (CD11b, an immune cell receptor that blocks extravasation of immune cells; Williams et al., 2019). The results identified new monocyte-related inflammatory pathways and blocking these pathways reduced monocyte infiltration and provided neuroprotection in DBA/2 J mice.

The anti-inflammatory effects of Ibudilast, a cAMP phosphodiesterase inhibitor, were also investigated in a rat model of ocular hypertension (Vargas et al., 2016). Intraocular administration of Ibudilast reduced macroglia and microglia reactivity in the retina and optic nerve, thereby decreasing the amount of pro-inflammatory cytokines secreted. The study also showed that elevated IOP increased A-type PDE4 activity in Müller cells, and Ibudilast administration resulted in an increase in cellular cAMP. The combined injection of Ibudilast and Rp-cAMP (a non-hydrolysable analog of cAMP that inhibits protein kinase A) resulted in complete blockade of Ibudilast-induced neuroprotection. These results indicate that Ibudilast may be a well-tolerated and safe modulator of neuroinflammation in glaucoma, and that PDE4 represents a potential target for research into inhibiting chronic gliosis and neuroinflammation in glaucoma (Rolle et al., 2021).

Another pathway involved in neuroinflammation represents Toll-like receptors (TLR) signaling, particularly TLR2 and TLR4 present in microglia. Studies have shown that these pathways are involved in the transmission of signals induced by heat shock proteins and are associated with oxidative stress (Luo et al., 2010). It has also been observed that Tenascin C, which is one of the ligands for TLR4, undergoes increased expression both in human glaucoma patients and in animal models (Johnson et al., 2007; Wallace et al., 2015). TLRs are therefore an interesting target for research on inhibition and reduction of neuroinflammation. There are studies intensively using human umbilical cord mesenchymal stem cells (hUC-MSCs) to show their immunomodulatory properties (Cislo-Pakuluk and Marycz, 2017). These cells show the ability to regulate the function of microglia and astrocytes (Jose et al., 2014; Wang et al., 2018). They have also been used in studies to inhibit the activity of TLRs, with a positive effect in reduced neuroinflammation in a rat model of glaucoma (Lund et al., 2009; Zwart et al., 2009; Ji et al., 2018).

The NLRP3 inflammasome, a complex of multiple proteins, plays a crucial role in initiating inflammatory responses. Comprising NLRP3, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), and pro-caspase-1, this inflammasome is activated by various triggers. Upon activation, pro-caspase-1 undergoes cleavage into its active form, initiating the maturation and release of pro-inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-18 (IL-18; Chi et al., 2014).

Recent research has compellingly demonstrated the activation of the NLRP3 pathway in different types of glaucoma. Elevated levels of NLRP3 inflammasome components have been identified in the trabecular meshwork, retina, and optic nerve head of eyes affected by glaucoma (Ward et al., 2019). This upregulation of NLRP3 is closely associated with neuroinflammation and the death of retinal ganglion cells (RGCs), contributing to the progressive nature of glaucomatous optic neuropathy. A noteworthy connection has been suggested between the activation of the NLRP3 inflammasome and increased intraocular pressure (IOP). Mechanical stress, a common factor in glaucoma, may serve as a trigger for NLRP3 inflammasome activation, resulting in the release of pro-inflammatory cytokines. This cascade of events is implicated in the perpetuation of neuroinflammation and the subsequent damage to the optic nerve. The potential therapeutic implications of targeting the NLRP3 pathway in glaucoma are promising. Inhibiting NLRP3 activation, whether through pharmacological agents or gene therapies, has demonstrated neuroprotective effects in experimental models (Coll et al., 2015; Gong et al., 2019; Zhang et al., 2019; Coyle et al., 2021). However, further research is necessary to thoroughly explore the safety and efficacy of these interventions, paving the way for potential translation into clinical applications.

Oxidative stress is an imbalance between free radicals and antioxidant scavengers that can result in tissue damage (Pickering et al., 2013). Reactive oxygen species (ROS) are free radicals that are the main source of oxidative stress formation. These include superoxide anion (O₂⁻), lipid radical, hydroxyl radical and nitric oxide (NO). Uncontrolled production of ROS leads to oxidation of biological molecules such as proteins, lipids and DNA contained in the cell, which triggers activation of mechanisms leading to cell death by apoptosis or necrosis (Garza-Lombó et al., 2020). There are several mechanisms leading to ROS production, and the most important include uncoupled NO synthase, mitochondrial dysfunction, xanthine oxidase and NADPH oxidase (Dalle-Donne et al., 2006). Mechanisms related to oxidative stress are considered an important factor in the development of neurodegenerative diseases, including glaucoma (Levkovitch-Verbin et al., 2000; Himori et al., 2013; Lin and Kuang, 2014; Naguib et al., 2021; Sanz-Morello et al., 2021).

Autophagy is a lysosomal degradation of the cellular constituents. Cellular homeostasis is maintained by removing damaged organelles, such as mitochondria, in the autophagic process of mitophagy (Terman et al., 2006). However, intralysosomal accumulation of waste material increases with age. Oxidation of this material leads to the formation of a non-degradable substance called lipofuscin (Grune et al., 2005). Lipofuscin has been shown to accumulate in glaucomatous human lamina cribrosa cells. This accumulation may in turn alter cellular authophagic activity. In addition, autophagy markers have been found in lamina cribrosa cells (McElnea et al., 2011). Furthermore, studies using DBA/2 J mice showed that in myelinated optic nerve axons the number of mitochondria was increased, and they had significantly smaller surface area. These results indicate that mitochondrial pathology may be the cause of the energy deficit in glaucomatous optic nerve. An increased number of autophagosomes was observed, which, together with increased number of mitochondria and decreased Lamp1 (lysosomal associated membrane protein 1) gene, suggests that deteriorating mitochondria do not recycle efficiently enough in the mitophagy process (Coughlin et al., 2015). These findings suggest that mitochondrial dysfunction and the resulting oxidative stress may be an important factor in the pathogenesis of glaucoma.

In addition to the well-known correlation between mitochondrial dysfunction and oxidative stress, several recent studies have pointed to a significant role for NADPH oxidase (NOX) in oxidative stress during glaucoma. NOX has been identified as a major source of oxidative stress in many retinal eye diseases, such as age-related macular degeneration and ischemic retinopathy (Ruan et al., 2020). Reactive oxygen species produced by NOX affect the regulation of various cellular processes such as proliferation, differentiation, and migration (Chan et al., 2009). There are seven NOX isoforms, three of which are the most studied in the eye pathology: NOX1, NOX2 and NOX4. NOX1 and NOX2 produce superoxide, while NOX4 mainly produce hydrogen superoxide (Griendling et al., 2000). The enzymatic activity of NOX can be induced by various stimuli: hypoxia (Dvoriantchikova et al., 2012), proinflammatory cytokines, i.e., TNF-α (Geng et al., 2020) or heat shock proteins (Sakai et al., 2003). All of these stimuli are associated with glaucoma pathology. There are many studies using animal glaucoma models that show the relationship between NOX activity and pathological features (Yokota et al., 2011; Dvoriantchikova et al., 2012). Recently, many studies have focused on using pharmacological inhibition to block the contribution of NADPH oxidase in an animal model of retinal pathological conditions, such as ischemic retinopathy and retinal inflammation. However, there are very limited studies on the pharmacological inhibition of NOX in animal glaucoma models. In the ischemia reperfusion (I/R) injury model, the role of NOX2 in neuronal cell death was also examined (Yokota et al., 2011). The results show that I/R induces the retinal cell death, indicated by numerous TUNEL-positive cells in the inner retina and significant decrease in neuronal cell density. This effect was abolished in mice with Nox2 gene deletion, indicating a role for NOX2 NADPH oxidase in I/R-induces injury of the inner retina. Furthermore, Apocynin, a non-selective inhibitor of NADPH oxidase, was used in the study. Apocynin improved retinal morphology in NOX2^−/− with I/R injury, indicating the need for studies using other selective NOX inhibitors in glaucoma models.

Glutamate plays an important function in regulating neurophysiological processes. However, increased stimulation of relevant receptors with glutamate leads to neuronal damage and death. A similar mechanism takes place in the eye. Glutamate is secreted by ganglion cells, bipolar cells, and photoreceptors in response to light stimuli. Its homeostasis is ensured by the activity of microglia and macroglia cells. When glutamate homeostasis is disrupted, its uncontrolled increased concentration leads to excitotoxicity (Harada et al., 2007). Glutamate binds to the N-methyl-D-aspartic acid receptor (NMDAR), affecting the disruption of K⁺/Na⁺ ion homeostasis—there is an increased influx of Na⁺ ions into cells, resulting in the release of calcium ions, activation of the ERK 1/2 signaling pathway, formation of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction (Arundine and Tymianski, 2003). All these factors lead to apoptotic death of retinal cells. Intravitreal injection of glutamate in rats resulted in loss of RGCs, which is considered a major cause of vision loss in glaucoma (Lam et al., 1999).

There are attempts to interfere with the mechanisms of excitotoxicity in ischemic models. The first idea was the use NMDA receptor antagonists in a rat model of retinal ischemia, but its complete blockade affects other physiological processes of nerve cells (Gómez-Vicente et al., 2015). Another approach was the use of brimonidine, a selective α₂-adrenergic agonist that shows the ability to lower IOP and prevent the excitotoxicity in RGCs. Its proven mechanism of action is based on lowering glutamate secretion, regulating Ca²⁺ ion homeostasis, reducing NMDA receptor expression, or decreasing production of aqueous humor by the ciliary body (Lee et al., 2012; Villanueva et al., 2020). In vivo studies showed that intrathecal injection of brimonidine prevented the death of RCGs in a rabbit model with NMDA-induced excitotoxicity and in rat model of high-tension glaucoma (Dong et al., 2008; Ozdemir et al., 2009).

Other studies use glutamate transporters found in retinal cells as targets. One of these, called GLAST, found mainly in Müller cells, removes most glutamate molecules from the extracellular space (Vorwerk et al., 1999). Studies using GLAST knock-out mice have shown that about 50% of glutamate is captured by GLAST (Shi et al., 1997). Other studies indicate that increased IOP in animal models results in decreased GLAST expression (Schuettauf et al., 2007) and knocking out Glast gene leads to spontaneous degeneration of RGCs in normal tension conditions (Harada et al., 2010). Increasing glutamate transport is therefore an interesting target for studies on reducing pressure-induced neurodegeneration. There are studies in which the estrogen modulator, Tamoxifen, was used and showed neuroprotective properties due to up-regulation of GLAST, which reduced excitotoxicity (Lee et al., 2009). In a rat model of glaucoma, eye drops containing 17β-Estradiol were also used, which had the effect of reducing neurodegeneration (Prokai-Tatrai et al., 2013). Estradiol and its analogs are therefore interesting targets capable of increasing GLAST expression, which could be exploited in glaucoma patients.

Recent research has linked ATP binding cassette transporter A1 (ABCA1) to POAG. ABCA1 is a member of a superfamily of ABC transmembrane transporters responsible for removing cholesterol by transferring it to lipid-free apolipoprotein A-I and apolipoprotein E (Danik et al., 1999). Its transcription is regulated by liver X receptor (LXR), a member of the nuclear receptors superfamily (Yang et al., 2011). ABCA1 is prominently expressed in various ocular tissues including the trabecular meshwork, Schlemm’s canal, endothelial cells, optic nerve, and retina, particularly in retinal ganglion cells (Cai et al., 2015). Recent studies suggest ABCA1’s potential role in regulating intraocular pressure (IOP), although the precise mechanism remains unclear. Hu et al. propose a connection between ABCA1-mediated IOP regulation and the Cav1/eNOS/NO signaling pathway, which is implicated in glaucoma pathogenesis in POAG patients. ABCA1 interacts with caveolin-1 (Cav1), a key player in cellular processes like vesicular transport, cholesterol regulation, and mechanosignal transmission (Kuo et al., 2011; Gu et al., 2017). Cav1 regulates vasodilation by modulating endothelial nitric oxide synthase (eNOS), known as a pressure-dependent regulator of IOP through the conventional outflow tract pathway (Daniel Stamer et al., 2011). ABCA1 expression levels were examined in samples of trabecular meshwork from POAG patients and human donor eyes. ABCA1 expression levels were analyzed in samples of trabecular meshwork from POAG patients and human donor eyes, showing higher expression in POAG patients compared to controls. Studies on cells and animal models suggest that ABCA1 can modulate Cav1 expression, alleviating Cav1’s inhibitory effect on eNOS, thus increasing eNOS-derived production and reducing IOP (Hu et al., 2020). Based on these findings, enhancing the ABCA1 signaling pathway is proposed as a potential therapeutic approach for glaucoma and ocular hypertension treatment.

The Rho kinase (ROCK) activation pathway is a critical signaling pathway involved in various cellular processes such as cell migration, proliferation, adhesion, and smooth muscle contraction (Thumkeo et al., 2013). Rho-associated protein kinases (ROCK) are a serine/threonine protein kinases, that are downstream effectors of small GTPase Rho. The activation process involves the binding of Rho-GTP to the kinase domain of ROCK, resulting in a conformational change that enhances ROCK activity. Activated ROCK phosphorylates various downstream substrates, including myosin light chain (MLC), myosin phosphatase substrate 1 (MYPT1), kinase C-potentiated phosphatase inhibitor 17 (CP1-17), LIM kinase (LIMK), calmodulin (CaM), ezrin, radixin, and moesin. Through these interactions, the Rho-ROCK signaling pathway orchestrates the dynamics of actin cytoskeleton, contraction mediated by actomyosin, adherence between cells, cellular morphology, and the reorganization of the extracellular matrix (ECM; Amano et al., 2010). Researchers have indicated that ROCK inhibitors have the capability to influence trabecular meshwork, enhancing the drainage of aqueous humor from the eye and subsequently decreasing IOP (Honjo and Tanihara, 2018). Currently, there are several ROCK inhibitors in use as a novel class of drugs that can act directly on trabecular meshwork. Three of them (ripasudil, netarsudil and fasudil) are commercially used in the treatment of glaucoma in some countries. Two of the ROCK inhibitors have not been approved yet (SNJ-1656—phase II clinical trial, Y-27632—pre-clinical phase; Pagano et al., 2023). With the rise in the use of ROCK inhibitors by ophthalmologists, it becomes important to enhance our comprehension of the potential advantages and safety profiles associated with these pharmaceuticals, whether employed in approved or off-label capacities.

Purinergic P2 receptors binds extracellular nucleotides. The superfamily contains two types of receptors. First, G protein-coupled P2Y binds ATP, ADP, UTP, UDP, UDP-sugars and interacts with G proteins inducting intracellular signaling pathways, and ionotropic P2X, which binds ATP and has two or three transmembrane domains and form an ion channel gating Na⁺, K⁺, and Ca²⁺ (Shinozaki et al., 2023). Normally, the level of extracellular ATP is extremely low. ATP levels can rise due to uncontrolled release from necrotic cells or controlled release via exocytosis or channel-mediated mechanisms. Ocular tissues like the lens, retina, corneal endothelial cells, ciliary body, and retinal astrocytes are known to release ATP and nucleotides. There are indications that a sudden increase in intraocular pressure (IOP) prompts ATP release from the retina. Within the dynamics of aqueous humor, an increase in ATP or UTP prompts the activation of P2Y₂ receptors, resulting in an escalation of aqueous humor production. On the other hand, when there’s less ADP/UDP, it stops the receptors P2Y_1/6 from slowing down or speeding up fluid production or drainage (Shevozaki et al., 2005; Taguchi et al., 2016; Hamada et al., 2021). These changes make the IOP go up, which could harm the RGCs. Disrupted purinergic signaling may damage RGCs through autonomous and non-autonomous pathways. P2X7 receptors, found in RGCs, microglia, astrocytes, and Müller cells, play an important role. RGCs experience excitotoxicity when these receptors are activated. In glial cells, activation leads to neurotoxic responses, including microglia releasing pro-inflammatory cytokines and ROS that harm RGCs. Astrocytes release glutamate upon activation, overstimulating glutamate receptors in RGCs, while Müller cells reduce glutamate uptake, further damaging RGCs. Additionally, P2X₇ receptor activation in Müller cells triggers inflammation.

Shevozaki et al. (2005) showed that P2Y₆ lowers IOP – ablation of the P2Y₆ gene in mice (P2Y6 KO) results in hypertensive glaucoma–like optic neuropathy—exhibited sustained elevation of IOP, age-dependent damage to the optic nerve, thinning of ganglion cell inner layers, and a reduction of RGC numbers. Topically applied uridine diphosphate, an endogenous selective agonist for the P2Y₆ receptor, decreases IOP. In that case P2 receptor ligands may be a promising candidate for glaucoma treatment.