The experimental autoimmune uveitis (EAU) model is a noninfectious uveitis animal model that closely resembles human uveitis in both clinical and histological features (49-51). EAU in mice is induced through the subcutaneous injection of an emulsified antigen, which disrupts immune tolerance within the body. Following immunisation, naïve T cells receive antigens delivered by presenting cells. Subsequently, these cells are converted into Th0 cells, and T-cell subpopulations such as Th1 and Th17 differentiated from these cells have important functions in numerous autoimmune diseases, including EAU (52,53). These T cells multiply in the peripheral system and translocate to the retina, where they release inflammatory factors and promote macrophage migration, causing tissue damage (50).

Diverse immune-cell infiltration is a hallmark of the EAU retina, involving macrophages, neutrophils, dendritic cells and other immune cells (54). Furthermore, the proportions of various immune cells were observed to vary across different phases of EAU. During the acute EAU phase, macrophages accounted for 40% of all retinal immune cells. However, in the late chronic stage, the percentage decreased to 19%. By contrast, the percentage of immune cells, such as CD8 T cells and myeloid-derived suppressor cells (MDSCs), increased during the transition from the acute to the chronic phase (54).

In addition, the phenotypes of immune constituents infiltrating different EAU stages undergo dynamic changes. For instance, in the acute phase, most macrophages exhibit the M1 phenotype, whereas in the chronic (angiogenic) phase, M2 macrophages are predominant (55). These results suggest that the retinal microenvironment under inflammatory conditions determines the subsets of infiltrating cells, in addition to controlling the phenotypes of different types of immune cell.

Studies have underscored the involvement of innate immune cells, particularly macrophages and microglia, in antigen presentation in EAU (56). Macrophages are recognised as crucial effector cells in EAU and contribute to the inflammatory process by releasing inflammatory cytokines (57). Retinal microglia exhibit phagocytic and pathogenic characteristics similar to those of macrophages. Upon activation, both macrophages and retinal microglia release pathogenic factors such as TNF-α and iNOS, resulting in the nitration of cytochrome c, which is known to cause EAU-cell apoptosis (58-60).

The aryl hydrocarbon receptor (AhR), a high-molecular-weight transcription factor, has been demonstrated to exert a negative regulatory effect on LPS-mediated inflammatory responses in macrophages (61). This suggests that AhR may be involved in the negative regulation of M1 polarisation. After EAU induction, AhR-/mice had more severe clinical and histopathological manifestations of uveitis than AhR mice. Compared with AhR EAU mice, AhR+/+−/− EAU mice showed evidence of a significant increase in macrophages/microglia and a greater polarisation of phenotypes from M2 to M1 (62).

Furthermore, research has shown that the use of 2,3,7,8-tetrachlorodibenzo-p-dioxin (an AhR activator) can activate AhR through the NF-κB, STAT1 and STAT3 signalling pathways. This induces macrophage M2 polarisation, reducing the production of apoptotic cells and the release of pro-inflammatory factors. Consequently, the clinical manifestations of EAU are alleviated (62).

IL-33 is a member of the IL-1 cytokine family and signals through a heterodimeric receptor composed of suppression of tumorigenicity 2 (ST2) and IL-1R accessory protein (63). Studies have highlighted the crucial role of the IL-33/ST2 pathway in enhancing the polarisation of alternatively activated macrophages (M2) (64).

Following 21 days of EAU induction, ST2-deficient mice showed worse clinical symptoms than non-knockout mice, whereas treatment of wild-type (WT) mice with IL-33 significantly improved uveitis lesions. This improvement was accompanied by a significant increase in the proportion of CD206 and CD273 cells, suggesting that the upregulation of the IL-33/ST2 signalling pathway drives macrophage (M2) polarisation, thus attenuating the clinical manifestations of EAU (65).

Furthermore, glucocorticoids have been reported to mediate the P38-MAPK/myocyte enhancer factor-2c axis, thereby promoting the polarisation transition of macrophages from M2 to M1 and the release of anti-inflammatory factors. Consequently, this process inhibits EAU and fosters the healing of damaged eye tissue (66).

Suppressor of cytokine signalling (SOCS) proteins, particularly SOCS1 and SOCS3, regulate macrophage polarisation and cytokine expression. For instance, in BMDMs, SOCS3 acts as a negative regulator of GM-CSF-induced expression of CCL2, Arg-1 and matrix metallopeptidase 12 (67,68). LysM^Cre/+SOCS3^fl/fl mice (LysM^Cre/+SOCS3^fl/fl mice were obtained by crossing SOCS3^fl/fl mice with LysM-Cre mice, a type of mouse with SOCS3 deficiency in myeloid cells) showed an increased proportion of GM-CSF in the intraretinal milieu, which may have triggered the release of CCL2 and Arg-1 from macrophages.

Research has demonstrated that mice deficient in SOCS3 (LysM^Cre/+SOCS3^{f l/f l}) experience enhanced retinal degeneration and accelerated retinal angiogenesis owing to inflammation (69). In the acute phase of EAU, LysM^Cre/+SOCS3^fl/fl mice exhibited increased numbers of infiltrating neutrophils and decreased numbers of macrophages compared to WT mice. Real-time reverse transcription PCR analysis revealed a significant upregulation in the release of TNF-α, IL-1β, IFN-γ, GM-CSF and Arg-1 in the retina of LysM^Cre/+SOCS3^fl/fl mice compared to that in WT mice. Furthermore, the percentage of Arg-1+ infiltrating cells was notably higher in LysM^Cre/+SOCS3^fl/fl EAU retinas than in WT EAU retinas. In the absence of SOCS3, both macrophages and neutrophils expressed higher levels of Arg-1, CCL2, IL-6 and VEGF, promoting angiogenesis, suggesting that deletion of SOCS3 partially induced M2 polarisation. Both isoforms of arginase have been implicated in vascular cell dysfunction and vessel wall remodelling in various diseases (70). Arg-1 is a characteristic marker of M2-type macrophages. To treat EAU, researchers utilised an Arg inhibitor, amino-2-borono-6-hexanoic acid, which effectively inhibits retinal angiogenesis without improving inflammation (69). This suggests that the development of retinal fibrovascular membranes in EAU is associated with the polarisation of macrophages to the M2 phenotype.

Optic neuritis, an acute inflammatory demyelinating disease of the optic nerve, is an initial symptom of multiple sclerosis (MS). It is characterised by optic nerve degeneration and loss of retinal ganglion cells (RGCs), resulting in permanent visual impairment; however, reliable treatments for this condition are currently lacking. The well-established experimental autoimmune encephalomyelitis (EAE) mouse model used for studying MS has also proven useful for investigating optic neuritis. The mouse model is characterised by the upregulation of molecules involved in inflammation, gliosis and macrophage infiltration (71). Accumulation of inflammatory factors leads to macrophage infiltration, which subsequently produces a large number of potentially harmful cytokines, further fuelling the inflammatory process (72).

EAE, triggered primarily by autoimmune Th1 and Th17 cells, is an inflammatory disease of the CNS (73). These cells produce various cytokines, including IFN-γ, TNF and GM-CSF, which participate in the M1 polarisation process of macrophages. Approximately 70% of the immune cells in the inner environment of the CNS in an inflammatory state are macrophages that are responsible for most neuronal tissue damage by releasing TNF, NO and other inflammatory factors (74-77). During EAE pathology, macrophages exhibit a dual-activated phenotype that expresses both M1 and M2 markers, such as CD86 and chitinase-like protein 3 (78). During EAE, both M1 (IFN-γ) and M2 (IL-4) cytokines are present in the inflamed CNS. Therefore, promoting the conversion of M1 subpopulation macrophages to the M2 subpopulation can promote the repair of MS-related damage and ameliorate functional impairment (6). Studies have demonstrated that fatty acids (FAs) have a positive impact on neuronal rescue by modulating macrophage phenotypes, reducing pro-inflammatory capacity and enhancing tissue recovery capacity (79).

Among M1-related factors, IL-12 and IL-23 are largely involved in the progression of EAE (80) by inducing macrophage recruitment through the upregulation of CXCL-10 and CXCL-11 release (81). Conversely, M2-related markers, such as CCL-2, promote the repair of neuronal axons in the EAE model (82), and CCL-22 upregulates the migration of anti-inflammatory immune cells during EAE progression (83). Furthermore, studies have demonstrated that treatment with ω-3 FAs reduces RGC damage by regulating the conversion of the M1 to the M2 subpopulation (84).

In the context of optic neuritis in an EAE model, suppressing M1 subpopulations and activating M2 subpopulations can effectively prevent retinal inflammatory processes (85). This intervention holds promise for hindering optic nerve damage and protecting RGCs from death.

SO is a type of uveitis characterised by granulomatous lesions that occur after ocular surgery or penetrating trauma (86). It appears to occur as a delayed-type hypersensitivity reaction to antigens in tissues exposed to traumatic events (87,88). The histopathology of SO is often characterised by choroidal capillary involvement, the presence of eosinophils, inflammation within the scleral canal, and substantial infiltration by B lymphocytes and macrophages (89,90).

To further investigate the role of macrophages in the inflammatory process of SO, researchers have performed immunohistochemical staining of choroid tissue obtained from patients clinically diagnosed with SO. Their analysis revealed a significant presence of infiltrating CD68 cells, along with the infiltration of TNF-α, INF-γ and other cytokines (91). These findings strongly suggest that macrophages are involved in the pathological processes underlying SO.

The presence of Dalen-Fuchs nodules suggests granulomatous inflammation in the middle of the retinal pigment epithelial (RPE) and Bruch's zones (88,89,92). Granulomas primarily consist of activated macrophages (93,94) and may arise within the retina. Studies have shown that M1 macrophage-specific cytokines, such as IL-23 and CCL19, account for a large proportion of granulation tissue in SO (93), suggesting that most inflammatory cells in SO Dalen-Fuchs nodules and granulation tissue are M1 macrophages.

RP is a retinal degenerative disease accompanied by the apoptosis of photoreceptor cells, often leading to severe visual impairment. Degeneration of photoreceptors is initiated by microglial activation, infiltration of macrophages, and accumulation of immunoglobulins and complement factors, resulting in persistent inflammation, proliferation of macroglial cells and progressive apoptosis of retinal neurons (95,96). Consequently, it is crucial to explore avenues for RP intervention therapy that involve the regulation of microglial activation and suppression of the inflammatory response.

As RP advances, the blood-retinal barrier becomes disrupted, leading to the recruitment of macrophages into the retina. This recruitment has an important role in activating immune cells and triggering the release of pro-inflammatory factors, which further exacerbates disease progression and ultimately leads to the loss of the retinal photoreceptor layer (97). Blood-borne immune cells have a significant role in the microenvironment associated with RP and are considered key mediators of the development of neurodegenerative diseases (98,99).

Resident microglia and invading macrophages coordinate responses to CNS injury by restoring tissue loss and causing neuroinflammation (14,100). These immune cells have distinct phenotypes, such as M1 macrophages that foster inflammation and M2 macrophages that facilitate tissue repair and regeneration (101). Given the contrasting roles of these macrophage subsets, recent therapeutic approaches to nervous system inflammation are being tuned from immune cell suppression to achieving a balance through molecules that regulate the M1/M2 phenotypic polarisation switch (102).

Studies have demonstrated that olfactory ensheathing cell transplantation holds promise for regulating the polarisation of retinal macrophages from M1 to M2 in Royal College of Surgeons rats via the JAK2/STAT3 pathway. This approach reduces the infiltration of activated M1 macrophages and fosters a less inflammatory microenvironment (103). Significant improvements in both the functional and structural aspects of vision can be achieved through this intervention. Similarly, essential FA supplementation improves retinal dysfunction and degeneration by reducing inflammation and microglial activation, weakening M1 markers, and inducing the transformation of rd10 mice (a model of autosomal recessive RP) retinas and LPS-stimulated BV10 cells to the M2 phenotype (104).

Glaucoma is a neurodegenerative disease characterised by optic nerve atrophy and irreversible loss of RGCs (105). Secondary degeneration of RGCs has a critical role in the progression of glaucomatous damage (106), as RGC apoptosis may continue even after intraocular pressure is reduced. Hence, delaying secondary RGC degeneration holds promise as a potential therapeutic approach for glaucoma treatment.

Damage to RGCs can be categorised as primary (resulting from direct injury to the axon or cell body, such as axonal extrusion or transection) or secondary (resulting from the release of toxic effectors from adjacent dying cells) (107-110). To investigate the secondary degeneration of RGCs, researchers have developed the partial optic nerve transection (PONT) model. In comparison with the complete optic nerve transection and optic nerve crush models, which damage all axons simultaneously, the PONT model offers an advantage, as it only damages a portion of the inner axons of the optic nerve, leaving others intact. This enables the separation of the primary from the secondary injury (111).

In glaucoma, the mechanisms leading to the death of RGCs are multifaceted and encompass the activation of microglia/macrophages, autophagy, disturbances in calcium regulation, apoptosis, oxidative stress, expression of pro-apoptotic proteins and neurotrophic deprivation (112). Microglia and macrophages have essential roles in inflammation, tissue restoration and homeostasis regulation following inflammation or CNS injury (113). Various subsets of macrophages contribute to the pro-inflammatory, anti-inflammatory, cell growth and tissue repair processes. Therefore, manipulating the activation state of microglia/macrophage subsets to foster favourable cytoprotection in response to injury may be a promising approach for glaucoma treatment.

The study revealed a significant increase in the release of CD68, iNOS and Arg-1 one week after PONT modelling, indicating an increase in M1 microglia/macrophages, which may contribute to RGC death. However, researchers found that polysaccharides extracted from Lycium barbarum could delay RGC degeneration by four weeks after PONT. This delay was accompanied by an increase in the number of activated microglia/macrophages and a higher count of M2-type microglia/macrophages. This suggests that L. barbarum regulates microglia/macrophage phagocytic activity and induces M2 polarisation, ultimately leading to delayed RGC damage (111).

Furthermore, in a glaucoma ganglion cell injury model (N-methyl-D-aspartic acid-induced retinal injury model), studies demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) increased the proportion of M2 subpopulations. In addition, PACAP promoted the release of factors such as TGF-β1 and IL-10 mRNA (112), both of which are markers of the M2 subtype of microglia/macrophages. The M2 subtype is associated with an acquired inactive state and is linked to reduced tissue damage, enhanced phagocytosis, increased synthesis of trophic factors, and decreased secretion of pro-inflammatory cytokines (25). These findings suggest that PACAP regulates the activation of microglia/macrophages toward the M2 subtype, thereby offering retinal protection against damage.

Clinically, non-arteritic anterior ischaemic optic neuropathy (NAION) often presents as optic disc oedema accompanied by acute painless visual loss (114). NAION is thought to arise from ischaemic damage to the optic nerve, which triggers an inflammatory response and oedema (115,116).

The optic nerve head is highly sensitive to changes in blood flow and is easily affected by factors such as autoregulation, vasospasm and systemic vascular diseases (117). In the context of NAION, inflammation is thought to be partially responsible for optic nerve damage (115,118). In a rat model of anterior ischaemic optic neuropathy (rAION), extracellular macrophages in the hypoxic region were recruited early and activated the resident microglia (119). Macrophages improve neuronal survival by secreting relevant factors and effectively phagocytosing myelin components to promote axonal regeneration (120). Furthermore, activated M2 microglia/macrophages have been linked to neuroprotection (121). However, it is important to acknowledge that in CNS diseases, activated microglia/macrophages may also release harmful factors, including pro-inflammatory cytokines and free radicals, which can cause damage to the nervous system (72). The proportion and subpopulation type of M2 microglia/macrophages in different pathological states may have distinct effects on the survival of RGCs and/or axon repair.

Research findings indicate that early treatment of the rAION model with G-CSF stabilises optic nerve vascular permeability, reduces macrophage recruitment near the optic nerve and induces M2 microglia/macrophage polarisation within the optic nerve (122). This treatment approach subsequently leads to a decreased expression of pro-inflammatory factors, prevents apoptosis induced by such factors and exerts neuroprotective effects in the rAION model.

Another study demonstrated that the binding complex of icariin and CCAAT enhancer-binding protein β significantly induces endogenous G-CSF expression by promoting alternative phosphorylation of IκB kinase-β, inhibitor of NF-κB (123). The elevated G-CSF expression then triggers noncanonical NF-κB activation, which further activates the PI3K/serine/threonine protein kinase B-a (AKT1) signalling pathway and promotes M2 microglia/macrophage polarisation, thereby preventing neuroinflammation and RGC apoptosis after optic nerve infarction in a rAION. In addition, ω-3 polyunsaturated FAs have also been found to possess neuroprotective effects in rAION by promoting the transformation of M1 macrophages into M2 macrophages. This transformation subsequently reduces the release of pro-inflammatory factors, such as TNF-a, iNOS and IL-1β, exerting anti-inflammatory effects that mitigate cytokine-induced optic nerve damage and help maintain RGC survival after infarction (84).

Furthermore, puerarin treatment has been found to stimulate the PI3K-AKT signalling pathway and sustain AKT1 activation, resulting in microglia/macrophages releasing CCAAT enhancer-binding protein β and hallmark M2 markers, such as Arg-1 and IL-10 (124,125). Polarisation of M1 microglia/macrophages into M2 microglia/macrophages reduces the proportion of TNF-α and IL-1β after optic nerve infarction, effectively preventing subsequent cytokine-induced optic nerve injury.

PVR is characterised by extensive proliferation and shrinkage of cell tissues at the posterior interface of the vitreous and inner surface of the retina (130). Shrinkage of these cell membranes can lead to traction retinal detachment or the reopening of previously treated retinal tears, resulting in severe visual impairment. Although the exact pathogenesis of PVR remains to be fully elucidated, the prevailing view is that it is a long-term injury repair process involving the activation of inflammatory cells, cytokine production, ocular cell proliferation and scarring (131).

A pivotal characteristic of PVR is the formation of myofibroblast membranes from transdifferentiated RPE cells and other cell types, including macrophages (132). Macrophages are considered one of the most crucial inflammatory cell types (133). By comparing vitreous samples from patients with PVR and uncomplicated retinal detachment, researchers have noted a significant increase in the number of monocytes/macrophages in vitreous samples from patients with PVR. Monocytes/macrophages were found to peak in the first 30 days after symptom onset in PVR and gradually decline over the subsequent three months (134). The abundance of macrophages in the intraocular microenvironment in the early stages of PVR and their sustained presence during progression underscore their vital role in the pathological process of PVR.

Macrophages have an irreplaceable role in the development of PVR through their ability to phagocytose damaged cells and tissues and release various growth factors and cytokines that mediate fibroblast chemotaxis and proliferation (135). Among the macrophage subsets, M2 macrophages, identified by the expression of Arg-1 and CD206, are particularly important in tissue repair and fibrogenesis (136).

In studies conducted in a rabbit model of PVR (137), researchers observed a swift onset of intense inflammation within the initial two weeks following PVR induction, with inflammation continuing to escalate until the 4-week mark post-induction. After the inflammatory phase, RPE cells undergo EMT, transitioning into fibroblast-like cells, which then give rise to contractile membranes. Throughout this process, the levels of growth factors, such as IFN-γ, VEGF, platelet-derived growth factor BB, placental growth factor and angiopoietin-2, surge, potentially fostering the survival, proliferation and EMT of RPE cells. The formation of contractile membranes and the secretion of growth factors are closely linked to M2 macrophages (133,138). Studies based on vitreous samples from human patients have indicated that M2 macrophage-derived microparticles can stimulate the proliferation and migration of RPE cells by activating the PI3K/AKT/mTOR signalling pathway (139), thereby contributing to the pathogenesis of vitreoretinal diseases.

Furthermore, studies based on vitreous samples from human patients suggested that M2 macrophages may contribute to the development of fibrovascular membranes in diabetic proliferative retinopathy (140). In a mouse model of PVR, CD206-positive M2 macrophages were found near the surface of the fibrous proliferation membrane, and the γ-secretase inhibitor DAPT was shown to inhibit RPE cell-induced PVR formation (decreased α-smooth muscle actin expression) and inhibit the infiltration of M2 macrophages by specifically targeting the Notch signalling pathway, thereby ameliorating PVR (141). These findings underscore the critical involvement of M2 macrophages in the pathogenesis of PVR and present a potential therapeutic target for intervention.

Choroidal neovascularisation (CNV) is the primary cause of vision loss in neovascular age-related macular degeneration (nAMD), with CNV possibly progressing to end-stage fibrous plaques and disc scarring (142). In nAMD, the accumulation of drusen may lead to reduced oxygen diffusion in the choriocapillary plexus, eventually culminating in CNV. The subsequent growth of new abnormal blood vessels in the subretinal space often results in haemorrhage, triggering a wound-healing response that eventually leads to subretinal fibrosis (126).

Fibrosis is a healing process that occurs in response to tissue injury (128). During the healing phase, angiogenesis is triggered to facilitate tissue repair, enhance oxygen supply and facilitate the migration of inflammatory cells to the lesion area (143). In nAMD, CNV develops in the subretinal and/or subpigmented epithelial spaces, causing haemorrhages and leaks that ultimately lead to subretinal fibrosis. This process involves the recruitment and/or migration of various cell types. These cells interact with inflammatory factors, causing significant remodelling of the ECM (144).

Histopathological examination of human eyes revealed significant recruitment of macrophages during CNV, where they have a role in the development of pathological neovascularisation, drusen formation and fibroblast scaffolds (145). Direct anatomical and functional evidence suggests that circulating macrophages rather than resident macrophages are responsible for laser-induced CNV. To construct subretinal fibrosis models, researchers commonly use a laser-induced acute wound-healing model of CNV (146,147). In this experimental setup, macrophages were found to promote the formation of fibroblast scaffolds during the early wound-healing response of laser-affected CNV lesions, with most macrophages at the laser injury sites being activated M2 macrophages (147). CNV membranes infiltrated by M2 macrophages were more susceptible to fibrosis than those with M1 macrophage infiltration (148).

Studies based on the laser-induced subretinal fibrosis model have demonstrated that inhibiting macrophage transition to the M2 subpopulation via the PI3K/Akt axis contributes to the improvement of fibrotic lesions in subretinal fibrosis (146). In addition, the application of triptolide has shown promise in reducing subretinal fibrosis by inhibiting the polarisation of M2 subpopulations and suppressing the activation of the TGF-β1/Smad axis, thereby downregulating TGF-β1-induced EMT/endothelial-MT (149).

Uveal melanoma is the most prevalent primary intraocular malignancy in adults, with an incidence of ~6-7 new cases per million individuals (151). The current treatment modalities include enucleation and radiation therapy. Although these treatments can curtail primary tumour growth, they remain ineffective in preventing tumour metastasis, leading to death within ~1-3 years (152).

Studies have provided compelling evidence for the pivotal role of macrophages in melanoma growth and survival. Melanoma-derived exosomes have also been identified as mediators of immunosuppression (153). These exosomes exert their effects by directly interacting with and suppressing various lymphocytes or by inducing MDSCs. In turn, MDSCs promote M2 subset transformation and recruit tumour-promoting regulatory T cells (154).

Researchers have postulated that lysing IFN-γ released by both tumour cells and immune cells in the microenvironment may be a contributing factor in transforming macrophages from a tumour-promoting M2 phenotype to an anti-tumour M1 phenotype, primarily through the IFN-γ/JAK-STAT1 pathway (155,156). In a mouse xenograft model, treatment with the oncolytic herpes simplex virus 1-enhanced green fluorescence protein through vitrectomy injection led to increased IFN-γ levels, an elevation in M1 macrophages and a reduction in M2 macrophages in peripheral blood, intraocular sites and distant tumours. In vitro experiments have further demonstrated a significant increase in IFN-γ at both the RNA and protein levels following oncolytic virus infection (157). Consequently, this treatment approach effectively reduced intraocular and subcutaneous tumours throughout the body.

However, it has been observed that melanoma exosomes can induce both M1 and M2 representative factors, namely TNF-α and IL-10, respectively (154). Furthermore, macrophage function assays have revealed an increasing trend from iNOS (M1) to Arg-1 (M2) activity, indicating that melanoma exosomes can induce a 'mixed' M1 and M2 tumour-promoting macrophage activation phenotype. Thus, in the pathological progression of uveal melanoma, M1 and M2 macrophage subpopulations seem to have flexible adaptability to tumour survival.

Diabetes is a metabolic disorder primarily characterised by hyperglycaemia stemming from abnormal insulin secretion and insulin resistance. Among patients with DR, microvascular complications are the most common manifestation. DR is categorised into non-proliferative DR (non-PDR) and PDR (PDR), with a distinction based on the presence of retinal neovascularisation (160). Non-PDR is typically characterised by asymptomatic microvascular changes, whereas PDR is involved in angiogenesis (161). DR is characterised by the abnormal growth and leakage of small blood vessels, leading to local oedema and associated tissue dysfunction. Dysregulation of vascular regeneration and inflammation are thought to be involved in the pathogenesis of DR (162,163).

Researchers have observed that upon high-glucose stimulation, microglia initially polarise toward the M2a phenotype, a response that initially mitigates tissue damage (164). However, as time progresses, there is an escalation in the production of M1 pro-inflammatory factors, accompanied by a decrease in the production of M2 anti-inflammatory factors, gradually shifting the macrophages toward the M2b phenotype. In advanced stages, microglia tend to exhibit an M1 phenotype with pronounced pro-inflammatory effects. In a rat model of streptozotocin-induced DR, there was an increase in M1 polarisation and a decrease in M2 polarisation, and microglia tended toward M1 polarisation with increasing glucose concentrations (165). The levels of iNOS (an M1 marker) and Arg-1 (an M2 marker) were higher in the retinas of db/db mice at five weeks of age. However, at eight weeks of age, iNOS levels continue to increase, whereas Arg-1 levels return to baseline (166).

It has been indicated that melatonin inhibits the excessive activation of microglia in the retina of diabetic rats by inhibiting the PI3K/AKT/Stat3/NF-κB signalling pathway, i.e., reducing the number of microglia cells and promoting their anti-inflammatory properties (167). It was speculated that this is related to melatonin promoting the transformation of microglia from pro-inflammatory (M1) to anti-inflammatory (M2) cells. Similarly, it was demonstrated that inhibiting M1 polarisation and promoting M2 polarisation of retinal microglia in DR rats through the TLR4/MyD88/NF-κB p65 pathway can effectively improve early DR (168).

ROP is characterised by impaired retinal blood vessel growth and development in preterm infants, which frequently leads to visual impairment and blindness (169). The pathological process of ROP is divided into two stages: An initial phase marked by delayed vascular growth after birth accompanied by vascular regression, followed by a second phase of hypoxia-induced pathological angiogenesis (170). An abnormal vascular state disrupts the inner retinal environment, thereby exacerbating the ischaemic state and leading to retinal leakage, scarring and eventually blindness. Although treatments such as laser and cryotherapy have improved ROP-related blindness, visual outcomes remain suboptimal for treated patients. Laser treatment has been successful in resolving most threshold ROP cases (171) and 100% of pre-threshold ROP cases (172). However, eyes treated with cryofixation or laser photocoagulation often manifest structural sequelae (171), underscoring the pressing need for preventive and less invasive therapeutic approaches.

Vascular abnormalities and inflammatory cell recruitment are primary contributors to the progression of abnormal retinal vascular diseases, including PDR and ROP (173). Studies have shown that macrophages have a role in promoting abnormal angiogenesis during pathological retinal vessel growth and that M1 and M2 subpopulations of macrophages are present in the intraretinal environment of ROP (174).

Studies have demonstrated that microglia/macrophages are activated after P12 once oxygen-induced retinopathy (OIR) models are established. M1 microglia/macrophages were observed in neovascular tufts located in the retina, starting at P12 and reaching their peak at P17 upon returning to normoxic conditions. At this time-point, the NF-κB/STAT3 axis is triggered, which results in an increased proportion of M1 microglia/macrophages and an enhanced proportion of TNF-α and IL-6. Consequently, the neovascular clusters exhibit a progressive increase in volume from P12 to P17. However, a shift to M2-type microglia/macrophage activity occurs from P17 onwards during the advanced stages of OIR. The IL-4/STAT6/PPAR-γ axis is triggered from P17 and reaches its maximum at P20, promoting M2 microglia/macrophage transformation. This, in turn, results in a decrease in inflammatory factors and regression of neovascular clusters (175).

Furthermore, investigations have revealed that cytokines TNF-α and VEGF, released by the M1 subpopulation, promote abnormal angiogenesis through interactions with endothelial cells. By contrast, M2 macrophages promote vascular anastomosis. The involvement of Notch1 signalling has been reported, although the exact secretory factors remain to be elucidated (176). These findings underscore the coordinated engagement of M1 and M2 macrophage subsets in guiding retinal neovascularisation.

Promoting macrophage transition from the M1 to the M2 phenotype during the pathological process of OIR is thought to have anti-angiogenic benefits. To investigate this, Marchetti et al (177) used human umbilical cord blood to obtain enriched progeny CD14(+) cell populations, which were then injected into the eyes of OIR mice. The results demonstrated that only CD14(+) cells polarised into M2-type macrophages could promote the normalisation of retinal vasculature and control pathological neovascularisation. Consequently, areas of vascular occlusion and associated tissue hypoxia were reduced. A separate study found that in the OIR retina, activated microglia/macrophages were predominantly of the M1 type rather than the M2 type. Treatment with ferulic acid was shown to decrease the proportion of iNOS+ microglia/macrophages while increasing the release of Arg-1, suggesting its potential to transform microglia/macrophages from the M1 type (expressing iNOS, CD86, IL-6 and TNF-α) to the M2 type (expressing Arg-1, IL-10 and CD206), thus exerting a strong anti-angiogenic effect (178).

It has been demonstrated that blocking the activation of NF-κB signalling can effectively promote the transformation of M1 macrophages into the M2 phenotype in OIR mice and subsequently reduce the number of neovascular clusters (179). In addition, IL-17A neutralisation attenuates ocular neovascularisation by increasing the proportion of M2 macrophages and downregulating the release of VEGF from M1 macrophages (180).

To explore macrophage polarisation in an OIR mouse model, researchers assessed the retina and found a significant increase in both M1and M2-like macrophages compared to normal controls. Both M1 and M2 macrophages exhibit a pro-angiogenic effect, promoting human umbilical vein endothelial cell (HUVEC) proliferation and contributing to retinal pathological neovascularisation (181). Similarly, Ma et al (174) investigated patients with advanced ROP and revealed a pro-angiogenic and pro-inflammatory microenvironment, with M1 macrophages predominant over M2.

In studies focusing on the role of pigment epithelium-derived factor, it was shown to inhibit macrophage polarisation in the retinas of an OIR mouse model through the regulation of adipose triglyceride lipase in the MAPK and Notch1 pathways. Specifically, pigment epithelium-derived factor suppressed the Notch1 and MAPK signalling pathways by inhibiting adipose triglyceride lipase, leading to a significant reduction in the release of iNOS and Arg-1, which are characteristic factors of M1 macrophages and M2 subpopulations, respectively. This ultimately resulted in a reduction in retinal neovascularisation (181).

Consequently, the role of macrophages in neovascularisation in OIR models remains a subject of debate, as both M1 and M2 macrophages may be involved. On the one hand, in the OIR model, retinal neovascularisation can manifest in two forms: Pathological neovascularisation, characterised by the emergence of abnormal blood vessels sprouting from the retinal surface into the vitreous, and physiological revascularisation, which involves the restoration of avascular regions with functional intraretinal vessels (182). Therefore, their proportions at different pathological stages may have contradictory effects. Ritter et al (183) found that a large number of migrating cells were localised in the retinal ischaemic area with a large loss of microglia, which may replace the function of microglia and promote vascular remodelling in the damaged area by releasing an appropriate amount of VEGF. Therefore, their location within the retina may also be one of the reasons for their contradictory roles in OIR models.

Wet AMD is a disease that causes vision loss due to the growth of CNV in the macula (184). Aberrant neovascularisation initially proliferates under the RPE band and then breaches the RPE band, causing intraocular haemorrhage and exudative serous retinal detachment, and later, discoid scarring (126). This localised loss of the retinal photoreceptor layer and RPE zone results in irreversible macular function loss and vision impairment.

CNV is considered involved in the submacular healing process (126,144). Angiogenesis has a crucial role in this process, and current clinical strategies predominantly focus on reducing the levels of VEGF, which is the primary factor that promotes angiogenesis (185,186). However, despite these efforts, only ~30% of patients with exudative AMD experience a three-line improvement in visual acuity, and ~15% of patients experience progressive deterioration, leading to legal blindness, even after receiving VEGF-inhibiting drugs (187-189). These results were expected, considering angiogenesis is an integral part of the complex healing phase. Hence, the search for alternative therapeutic approaches for CNV beyond anti-angiogenic treatments continues.

Multiple studies have explored AMD pathology and identified inflammation as a key driver of neovascular AMD progression (99,126,144,190,191). AMD is characterised by a chronic inflammatory response. Within this inflammatory milieu, macrophage recruitment and cytokine regulation are key mediators of CNV development (191).

Studies have indicated that macrophages are involved in abnormal angiogenesis in the pathology of CNV. M1 macrophages, characterised by specific markers such as iNOS, IL-6 and TNF-α, have been shown to inhibit angiogenesis (159). Conversely, M2 macrophages, identified using specific markers such as Arg-1, CD206 and CD163, promote pathological angiogenesis in CNV (192). Nakamura et al (193) revealed that increased IL-10 release in the eyes of aged mice activates associated signalling pathways, resulting in an increased proportion of M2 macrophages and the activation of vascular proliferative processes. Macrophage polarisation has emerged as a potential therapeutic target for CNV treatment.

In laser-impacted CNV, dynamic patterns of M1 macrophages and M2 subpopulations were observed, showing an early and immediate shift to M1, followed by a sustained shift to M2. M1 macrophages appear to be involved in the initial stages of CNV, whereas M2 macrophages have a critical role in the middle and late stages of CNV development and remodelling (194). For instance, during experimental CNV, upregulation of M1 signature factors (TNF-α and iNOS) was observed at day 3, suggesting inflammation at the onset of CNV lesions. By contrast, CD206 reached its maximum expression on day 7 of CNV formation, whereas CD86 and CD163 reached their maximum expression on day 14 of lesion formation. These marker genes represent different M2 macrophage subpopulations, suggesting that different macrophage subtypes have distinct roles at different time-points during the pathological process of CNV. Specifically, M2a macrophages may be associated with neovascularisation, whereas M2b and M2c macrophages may be involved in fibrous scarring (195).

CSF1, also known as macrophage CSF, has a crucial role in macrophage recruitment (196) and the transition to the M2 subpopulation (197). When the CSF1 receptor receives CSF1, the PI3K/AKT/forkhead box (FOX)O1 axis is activated, promoting M2 polarisation. Furthermore, under hypoxic conditions, HUVECs release more CSF1, thereby promoting macrophage migration and transition to the M2 subpopulation by upregulating the PI3K/AKT/FOXO1 axis. In a CSF1/CSF1 receptor (CSF1R)-associated manner, the M2 subpopulation upregulates the proliferation, recruitment and lumen formation of HUVECs. Inhibition of the CSF/CSFR axis has been shown to suppress M2 polarisation of macrophages and attenuate laser-induced CNV formation in mice (198).

In addition, studies have revealed that long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) promotes the expression of M2 macrophage markers by targeting phosphatase and tensin homolog via microRNA (miR)-148a-3p. Downregulation of NEAT1 can effectively inhibit CNV by suppressing the transformation of M2 macrophage subsets (199).

Researchers have proposed that the Rho-associated protein kinase (ROCK) signalling pathway is a key pathway in regulating macrophage polarisation, and that the expression of ROCK pathway-related factors and pathway signal transduction processes affect the pathological process of CNV. They conducted experiments by differentiating mouse BMDMs into M1 or M2 phenotypes and injecting them into the eyeballs of laser-modelled WT mice. They observed that CNV lesions were not altered by native-morphological macrophages but that M2 subpopulation macrophages promoted lesion progression, which was reversed in ROCK2 inhibitor-treated animals. By contrast, the M1 subpopulation ameliorated the damage caused by the CNV. Further intravitreal injection of the M1 subpopulation in laser-modelled mice treated with a ROCK2 inhibitor did not ameliorate CNV-induced damage, confirming that ROCK2 inhibits CNV lesions in vivo by promoting the polarisation transition of macrophages to M1 (200). Ras homolog family member A (RhoA) expression and myosin phosphatase target subunit 1 and myosin light chain phosphorylation are also upregulated in CNV and decreased by melatonin administration (201). The RhoA/ROCK axis promotes the transition of macrophages to the M2 subpopulation and prevents conversion of the M1 subpopulation, thereby triggering CNV lesions. Melatonin converts M2 microglia/macrophages to the M1 subset by inhibiting the RhoA/ROCK axis, resulting in the downregulation of CNV lesions, reduced associated vascular leakage and inhibition of abnormal vascular status in laser-affected CNV lesions.

Other studies have shown that miR-505 is abnormally upregulated in laser-induced CNV lesions. Transmembrane protein 229 B (TMEM229B) was identified as a direct target of miR-505-5p, and administration of an miR-505 inhibitor significantly upregulated the expression of endogenous TMEM229B in CNV mice. This specific inhibition of M2 polarisation in mice with CNV led to reduced VEGF expression and suppressed CNV formation. In vitro experiments further demonstrated that exogenous TMEM229B significantly inhibited the expression of the M2-specific markers Ym-1 and Arg-1 (202).

In addition, the IL-4 mutant protein IL-4/Q116E was found to regulate the inflammatory response of laser-induced CNV through the Notch/delta-like canonical Notch ligand 4/monocyte to macrophage differentiation-associated signalling pathway, increasing the expression of CD68 and CD80 and reducing the expression of Arg-1 in RPE choroidal tissue. Induction of macrophage polarisation from M2 to M1 attenuated CNV development (203).

Furthermore, in the context of a laser-induced CNV model, injured RPE upregulated 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3)-driven glycolysis in macrophages, resulting in the induction of hypoxia-inducible factor (HIF)-1α/HIF-2α and NF-κB. This subsequently induced the expression of macrophage subset-associated signature factors and pro-angiogenic factors, ultimately promoting the transformation of the M1 subpopulation of macrophages into the M2 subpopulation and promoting CNV development. However, the PFKFB3 inhibitor AZ67 effectively downregulated the expression of HIF-1α/HIF-2α and NF-κB signalling and largely prevented laser-affected CNV lesions (204).

Furthermore, researchers have used small interfering RNAs (siRs) to suppress TIMP metallopeptidase inhibitor 3 (TIMP-3) expression in BMDMs and RPE/choroidal tissues in a laser-induced mouse model of CNV. They found that the release of M2 biomarkers CD206, CD163, Arg-1 and Ym-1 was correspondingly upregulated in vitro and in vivo in the siR-TIMP-3 group, indicating that a lack of TIMP-3 may promote M2 macrophage differentiation. Furthermore, intraocular injection of siR-TIMP-3 was shown to upregulate the progression of CNV lesions, as detected using optical coherence tomography angiography, suggesting that TIMP-3 inhibition is associated with the M2 macrophage subset and has a key role in CNV formation (205).

Finally, studies have shown a greater proportion of M2 macrophages compared with the M1 subpopulation during three and seven days of buffer treatment in a laser-induced mouse model (206). Triptolide significantly downregulated the accumulation of the M2 subpopulation at the lesion site over the 3 and 7-day periods. Triptolide also reduced the proportion of M2 macrophages during the same periods. In addition, triptolide decreased the release of VEGF, intercellular adhesion molecule 1 and TNF-α in local CNV injury, consistent with a reduction in the total number of aggregated macrophages and a lower ratio of the M2 subpopulation. Consequently, intraperitoneal injection of triptolide inhibited the transformation of the M2 subpopulation in CNV focal lesion areas, resulting in the downregulation of inflammatory and angiogenic factors, thereby inhibiting CNV progression and macrophage infiltration in CNV focal areas (207).