Like other axons in the central nervous system (CNS), axons of the optic nerve have limited regenerative capacity due to the complex and inhibitory nature of their environment. Furthermore, axons in the optic nerve are highly specialized and need to regenerate in a precise manner, needing to reach out to both ipsilateral and contralateral eyes to restore vision, ultimately creating significant obstacles to achieving successful regeneration and functional recovery following damage. This stands in contrast to the axons of the peripheral nervous system (PNS), which are capable of successful regrowth after injury due to their more conducive environment.

Retinal ganglion cells (RGCs), found in the nerve fiber layer of the retina, form the axons of the optic nerve. They receive visual information from photoreceptors in the retina, form the optic nerve, decussate at the optic chiasm, and are the sole group of neurons responsible for conveying information to visual regions of the brain such as the lateral geniculate nucleus and superior colliculus (Figure 1) [1]. After optic nerve injury, RGCs may undergo axon loss and/or cell death, resulting in the irreversible loss of vision [2]. The rate and extent of RGC death varies depending on the type of injury as well as the distance from the eye [3]. To model optic neuropathy and glaucomatous injury, researchers utilize optic nerve crush (ONC) on experimental models. Only two weeks after ONC, the RGC population is significantly reduced, yet several resilient RGCs remain capable of regenerating axons [2]. It is worth emphasizing that there exist many different subtypes of RGCs, several of which have fundamentally distinct responses to injury and may play different roles in regeneration [3]. Overexpression of the transcription factor Sox11, for instance, induces death in alpha retinal ganglion cells (a-RGCs) but increases survival of intrinsically photosensitive retinal ganglion cells (ipRGCs) [3]. Nonetheless, the outcomes of ONC followed by axon regeneration help us to understand potential treatments that can be used to promote axon regrowth in the context of glaucoma and other neuropathies. For example, in primary open-angle glaucoma, RGCs typically lose axons, and residual RGC cell bodies survive for an extended time prior to any significant remodeling [4,5].

Understanding the inhibitory factors in the CNS environment is critical for developing strategies and treatments that promote axon regeneration. Under normal conditions, axon growth always accompanies the formation of a structure known as a growth cone (Figure 2a) [6]. However, following injury or damage to the CNS, microtubules, astrocytes, and other glial cells respond by forming scar tissue called a glial scar that forms both a physical and chemical barrier that inhibits axon growth (Figure 2b) [7]. Within the glial scar, reactive astrocytes produce and release chondroitin sulfate proteoglycans that restrict axon regeneration by interfering with the growth cone's ability to extend and navigate [7]. Furthermore, the extracellular matrix within the CNS, which influences cellular actions such as adhesion, migration, and signaling, possesses complex and inhibitory properties. Elevated calcium levels are also known to play a role in triggering signaling cascades spanning from axons to their cell bodies, ultimately resulting in long-lasting changes in affected neurons [8]. Another crucial aspect of the regenerative process involves the expansion of membranes through lipid insertion, and thus, comprehending the lipid profiles associated with regenerating axons may contribute to our understanding of the mechanisms driving axon growth [9].

Researchers are exploring various approaches, such as modifying the inhibitory environment through gene therapy, enhancing growth factors, and utilizing cell-based therapy techniques, to overcome the challenges of optic nerve axon regeneration (see Table 1) and improve the potential for successful regeneration and visual function.

Optic neuropathies, a term used to describe conditions in which the optic nerve is damaged, encompass diseases such as glaucoma, ischemic optic neuropathy, and optic neuritis. Although no therapies currently exist to cure these conditions, current clinical treatment options help to slow their progression.

Glaucoma, the leading cause of irreversible blindness worldwide, is a group of eye diseases that damage the optic nerve, often due to an increase in intraocular pressure (IOP) inside the eye. In the most common types of glaucoma, such as primary open-angle glaucoma and closed-angle glaucoma, the accumulation of IOP within the eye eventually puts pressure on the optic nerve, impeding its blood supply and causing a reduction in the flow of nutrients and oxygen to its nerve fibers [10]. Thus, current treatment options aim to reduce the IOP within the eye and prevent further damage to the optic nerve [10]. Normal tension glaucoma (NTG), however, is a form of glaucoma in which optic nerve damage and vision loss occur despite normal pressure within the eye. Treatment for NTG also aims to lower IOP but may additionally focus on improving blood flow to the optic nerve. Present therapeutic approaches for the management of glaucoma include oral medications, eye drops, laser therapy, and glaucoma surgery [10]. Glaucoma medications encompass a diverse range of drugs that work through various mechanisms, such as lowering the production of intraocular fluid or improving its outflow through the trabecular meshwork or uveoscleral pathway [10,11]. Laser treatments, such as selective laser trabeculoplasty and laser peripheral iridotomy, also focus on enhancing drainage and circulation of fluid within the eye [11]. If medications and laser treatments are not effective in controlling glaucoma, surgical options may be considered [10,11]. Common glaucoma surgeries include trabeculectomy, tube shunt implantation, and minimally invasive glaucoma surgery (MIGS) [10]. Ischemic optic neuropathy, which occurs when there is reduced blood flow to the optic nerve, can be categorized into two main types: anterior ischemic optic neuropathy (AION) and posterior ischemic optic neuropathy (PION) [12]. AION is the more common of the two and affects the anterior portion of the optic nerve, whereas PION affects the posterior portion of the optic nerve [12]. Because AION and PION are associated with systemic conditions such as abnormal blood pressure, diabetes, or giant cell arteritis, treating and controlling these conditions may help to slow down the progression of optic nerve damage [12]. Optic neuritis, an optic neuropathy most often associated with autoimmune diseases such as multiple sclerosis, is characterized by inflammation of the optic nerve [13]. Treatment for optic neuritis generally involves the managing the underlying cause of inflammation and alleviating symptoms through corticosteroids and pain medication [13].

It should be emphasized that these conditions manifest differently in each patient, and thus, treatment should be tailored to meet the needs of each individual. Close collaboration with an ophthalmologist is vital for patients with optic neuropathy to ensure appropriate treatment selection, timely adjustments, and ongoing monitoring of the condition's progression. As mentioned previously, these treatment approaches focus on halting the progression of disease and preventing additional loss of RGCs without necessarily guaranteeing vision restoration. Axons of the optic nerve must connect with their appropriate targets in the brain to successfully restore visual function. Therefore, comprehending the fundamental molecular mechanisms within the optic nerve environment is vital in uncovering successful treatment strategies for optic nerve regeneration and eventually facilitating vision recovery.

Over the past few decades, perhaps the most significant progressions in the field of optic nerve axon regeneration have revolved around the manipulation of genes via different signaling pathways. Specifically, transcription factors have high pharmacological potential due to their central role in cell signaling. Among the signaling pathways that have been demonstrated to promote robust regeneration are the Phosphatase and tensin homolog (PTEN)/mammalian target of rapamycin (mTOR) pathway, SOCS3/Janus kinase/signal transducers and activators of transcription (JAK/STAT3) pathway, KLF pathway, Sox11 pathway, and RhoA/Rho kinase (ROCK) pathway.

PTEN, which acts via suppression of the mTOR pathway, has emerged as one of the primary transcription factors that promote axon regeneration. The mTOR pathway functions in cell growth, protein synthesis, metabolism, and autophagy [ [14]]. In 2008, researchers in the Park lab uncovered that deletion of PTEN, which was shown to activate the mTOR pathway, results in significant neuronal survival and axon regeneration [15]. Tuberous sclerosis complex 1 (TSC1), a protein complex known to play a role in metabolic signaling and cellular stress, is also responsible for inactivation of the mTOR pathway [15,16]. TSC1 deletion alone was found to enhance axon regeneration and survival, though not as significant as PTEN [15]. Numerous additional research investigations have validated these findings, many of which incorporate PTEN deletion/mTOR activation in combination with other treatment modalities. In 2022, researchers investigated Elk-1, a transcription activator found downstream of PTEN [17]. Overexpression of Elk-1 in the presence of PTEN deletion enhances axon regeneration and RGC survival as its effects are inhibited by both PTEN and REST [17].

SOCS3 is a known cytokine signaling suppressor that acts on the JAK/STAT3 pathway by acting on gp130 [18]. Therefore, when deleted, SOCS3 leads to pathway activation and robust axon regeneration. Researchers discovered that when both PTEN and SOCS3 are deleted, there is a synergistic effect, leading to vigorous and sustained axon regeneration [19]. PTEN functions as a negative regulator of the mTOR pathway, while SOCS3 acts as a negative regulator of the JAK/STAT3 pathway. Additionally, c-myc, a protooncogene that experiences downregulation following axotomy, has been implicated as a key regulator of anabolic metabolism. Significant axon regeneration, extending beyond the optic chiasm, was observed when animals were treated with deletions of PTEN and SOCS3 alongside c-myc overexpression [19].

Kruppel-like factors (KLFs), which regulate axon growth ability in CNS neurons, are zinc finger proteins that bind to DNA elements and act as transcriptional activators or receptors [20]. At least 15 of 17 KLF family members are expressed in neurons, but researchers have identified that specifically the deletion of KLF4 and KLF9 promotes axon regeneration [20,21]. The Goldberg lab discovered that KLF4 knockout (KO) mice demonstrate increased axon regeneration but no effect on RGC survival following injury, and KLF9 KO promotes axon regeneration after ONC [21]. Dual-specificity phosphatase 14 (Dusp14), a gene target of KLF9, has also been shown to be crucial in KLF9's ability to suppress RGC axon growth ability via the activation of mitogen-activated protein kinases [22]. Although Dusp14 KO promoted RGC survival, no significant increase in axon regeneration was found [22]. The observed regeneration in KLF9 surpasses that of Dusp14 KO alone, underscoring the importance of delving into further investigations concerning the gene targets of KLF9 [22].

Reducing the expression of Sry-related high-mobility-box 11 (Sox11) has been shown to enhance axon regeneration after ONC. However, this downregulation also triggers the death of the alpha subset of RGCs [3]. Sox11 is a transcription factor that undergoes upregulation following ONC, and hence, downregulation of Sox11 induces axon growth and development. Downregulation of both Sox11 and PTEN resulted in a significant increase in RGC survival compared to PTEN KO only [23]. However, due to the inherent variations in RGC survival across subtypes, identifying specific subtype control of axon regeneration may be necessary for the development of future treatments involving Sox11.

ROCK is a serine/threonine kinase found downstream target of the small GTPase Rho. RhoA/ROCK signaling is closely related to the pathogenesis of several neurodegenerative CNS disorders and plays a role in neuronal functions including the growth, development, and migration of neurites [24]. Inhibition of the RhoA/ROCK pathway has shown to increase RGC density and axon regeneration via the mediation of myelin-associated axon growth inhibitors such as Nogo, myelin-associated glycoprotein, oligodendrocyte-myelin glycoprotein (OMgp), and repulsive guidance molecule [24]. In experimental models, inactivation of RhoA as well as intravitreal injection of ROCK inhibitors has shown to promote axonal growth in vivo following ONC [25, 26, 27].

These transcription factor pathways interact with each other and other molecular signaling pathways to orchestrate the complex process of optic nerve regeneration. Understanding their roles and interactions is crucial for developing strategies to promote axon growth, overcome inhibitory signals, and enhance functional recovery after optic nerve injury. Nevertheless, pharmacological use of transcription factors has many challenges including transcription factor purification, insignificant function protein concentrations in vivo, and limited target identification [28].

Neuroinflammation refers to the inflammatory response that occurs in the nervous system, including the optic nerve, in response to injury or damage. While inflammation is a normal part of the body's immune response to injury, excessive or prolonged neuroinflammation can have both beneficial and detrimental effects on optic nerve regeneration. Following optic nerve injury, immune cells such as microglia, macrophages, and infiltrating immune cells are activated and migrate to the site of injury [29,30]. These immune cells release pro-inflammatory cytokines including tumor necrosis factor-alpha, interleukin (IL)-1-beta, and IL-6, which have both beneficial and detrimental effects depending on the acute or chronic response [29].

During the acute inflammatory response following optic nerve injury, retinal microglia is the principal immune cells that are activated to promote tissue growth and protection [31]. After a prolonged chronic inflammatory response, retinal microglia releases excessive amounts of inflammatory mediators, thus inducing neurotoxic effects on RGCs and eventually contributing to RGC death [31]. Due to the significant role microglia play in retinal homeostasis, altering their function could potentially influence RGC death. Following ONC, mobile zinc (Zn2+), recognized for their ability to exacerbate microglial activation, accumulate within the amacrine cells [32]. Intravitreal injection of Zn2+ chelators, which effectively inactivate Zn2+, enables axon regeneration as well as RGC survival [32]. A recent study has also found that inhibition of aldose reductase (AR), an inflammatory mediator highly expressed in retinal microglia, protects RGC death [33]. AR catalyzes the rate limiting step of the polyol pathway of glucose metabolism and has been found to be involved with numerous inflammatory pathologies [34]. Administration of Sorbinil, an AR inhibitor, has been found to reduce RGC death, improve RGC function, and delay axon regeneration, likely by preventing cytokine secretion and thus preventing damage to other retinal cells [33].

In addition to the resident microglia released following optic nerve injury, macrophages have been identified as significant contributors in neuroinflammation. Oncomodulin (Ocm) is a calcium-binding protein that is secreted by macrophages and binds to RGCs with high affinity in a cAMP-dependent manner [35]. The Benowitz lab has established that Ocm serves as a powerful signaling molecule in the context of intraocular inflammation, and delivery of Ocm in various manners induces regeneration [30]. Following intraocular inflammation, macrophages also demonstrate a significant expression of a chemokine named stromal cell-derived factor 1 (SDF1) [36]. Via its cognate receptor CXCR4, SDF1 acts to enhance RGC survival and promote long-distance optic nerve regeneration, especially when combined with Ocm [36].

A conditioning lesion, a phenomenon in which a small injury is given to the lens prior to optic nerve injury, has been found to enhance RGC survival and axon regeneration in mammalian models. The concept of a conditioning lesion to the lens stems from the observation that neurons in the PNS possess a greater ability to regenerate compared to the CNS, and thus an injury to the lens, a non-CNS tissue, may promote a more favorable environment for optic nerve regeneration [37]. Lens injury (LI) to an intact optic nerve also triggers the inflammatory response, stimulating immune cells including macrophages, neutrophils, and Müller cells [38]. Conversely, injury to the optic nerve without any prior lens damage only leads to minimal activation of immune cells [38]. Research performed by the Benowitz lab showed that a LI prior to ONC leads to an 8-fold increase in RGC survival and a 100-fold increase in axon regeneration beyond the crush site [38]. Moreover, they discovered that a LI-induced conditioning lesion can be achieved through intravitreal injection of a yeast cell wall preparation called Zymosan [38]. Zymosan induces an inflammatory response similar to that of a conditioning lesion, which may play a role in clearing cellular debris and releasing substances that support axon regeneration [38]. Combining Zymosan injection with other treatments such as PTEN and cAMP have been found to have synergistic effects on axon regeneration, even inducing axon regeneration across the entire optic nerve [39]. Researchers have also attempted repeated lens injuries, which has demonstrated full-length optic nerve regeneration without the use of any genetic manipulations [37].

Neurotrophic factor (NF) signaling activation is another promising therapeutic approach for addressing neurodegeneration. NFs are a group of proteins involved with the development, differentiation, and survival of neurons [40]. After binding to specific receptors on the surface of neurons, NFs undergo retrograde transport to the cell soma where they initiate effects that promote cell survival [40]. Research has shown that after axon injury, RGC death occurs partly due to decreased retrograde transport of neurotrophic factors [41]. Thus, abnormalities in retrograde signaling could potentially underlie the pathophysiological mechanisms of various neurodegenerative diseases [41]. Among the NFs studied in the field of optic nerve regeneration are ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrophic factor (GDNF) [41]. CNTF is a neuropoietic cytokine belonging to the IL-6 family that activates the JAK/STAT and MAPK pathways and exacerbates the inflammatory response [42]. The administration of CNTF via different delivery methods such as adeno-associated virus (AAV)-mediated therapy, CNTF-chitosan, and bolus injection has demonstrated promising results with regards to RGC survival and axon regeneration [43, 44∗, 45]. CNTF administration coupled with GDNF has also been found to have synergistic protective effects on RGCs following optic nerve injury [46]. Additionally, AAV delivery of BDNF and CNTF stimulates axon regeneration and protection of RGCs following optic nerve injury [43]. In 2023, researchers made a significant discovery wherein CNTF-chitosan enabled the reconstruction of full-length regeneration as well as functional recovery of the adult rat visual system, an accomplishment that has rarely been reported before [44]. NFs play a role in promoting cell survival, enhancing axon growth and regeneration, modulating gene expression, inducing synaptic plasticity, and providing neuroprotective effects, and thus, the administration of exogenous or modulation of endogenous NFs may enable us to improve the outcomes of optic nerve injuries and degenerative diseases.

Stem cells, which are capable of differentiating into various specialized cell types, are extensively studied in the context of regeneration research, including the optic nerve. Stem cells are able to secrete various growth factors, cytokines, and extracellular vesicles that possess neuroprotective and immunomodulatory properties [47]. Cell-based therapy is designed to repair damaged cells in the body by altering the immune system and can be used to treat medical conditions in other areas of medicine, such as autoimmune and inflammatory disorders [47]. To promote the development of stem cells, researchers utilize specific culture conditions and signaling molecules, ultimately aiming to generate a population of functional retinal cells that can integrate into the damaged retina [48]. Recent research has shown that various types of stem cells, including mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), may be used to promote optic nerve regeneration in experimental models [48]. Nonetheless, numerous challenges persist, including the complexity of differentiation protocols, safety, and long-term viability of transplanted cells, and development of effective delivery methods.

Human MSCs derived from various regions of the body, including bone marrow and umbilical cord (HUMSC), have been found to have neuroprotective effects on the cornea, retina, and photoreceptor cells. MSCs have immunomodulatory functions and secrete a number of cytokines, including BDNF, CNTF, and GDNF [49]. Recent studies have shown that MSC treatment via intravitreal injection promotes RGC survival, long-term neuroprotection, and long-distance axon regeneration [50]. The positive effects of MSCs on RGC survival may also be attributed to the idea that iPSC-MSCs donate functional mitochondria to RGCs and protect against mitochondrial damage-induced RGC loss [51]. In 2022, a research study explored the therapeutic potential of HUMSC transplantation in a glaucomatous experimental rabbit model and found that transplantation significantly increases both axon and RGC regeneration as well as the retinal structure [52]. The application of ultrasound targeted microbubble destruction further improved the HUMSC distribution and resulted in more promising outcomes [52]. Mouse ESCs have also been studied via transplantation into the retina of RGC-ablated mice with successful differentiation to RGC lineage [53].

Fish and amphibians’ retinal Müller cells possess the remarkable ability to regenerate, whereas this capacity is significantly suppressed in mammals [54]. Therefore, another potential approach for regenerating RGCs involves the endogenous regeneration of retinal stem cells. Researchers have been developing different systems and methods to stimulate transdifferentiation from RGCs to Müller cells [55,56]. The axons originating from Müller cell-induced newborn RGCs are also competent in traversing the optic chiasm and extending to the visual centers of the brain, indicating that the newborn RGCs possess the typical characteristics of RGCs [48].