Murine models of neovascular AMD revisited: Mechanistic pathways, immune-metabolic crosstalk, and the impact of aging and sex

Chanel Harris; Mohammad A Nisar; Ronny Amamoo; Rahul Patel; Pamela M Martin; Ravirajsinh N Jadeja; Menaka C Thounaojam

doi:10.1016/j.exer.2026.111094

. Author manuscript; available in PMC: 2026 Oct 7.

Published in final edited form as: Exp Eye Res. 2026 May 29;270:111094. doi: 10.1016/j.exer.2026.111094

Murine models of neovascular AMD revisited: Mechanistic pathways, immune-metabolic crosstalk, and the impact of aging and sex

Chanel Harris ^a,¹, Mohammad A Nisar ^b,¹, Ronny Amamoo ^a, Rahul Patel ^b, Pamela M Martin ^a, Ravirajsinh N Jadeja ^a, Menaka C Thounaojam ^a,^b,^*

PMCID: PMC13639894 NIHMSID: NIHMS2210910 PMID: 42217724

Abstract

Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss in older adults. Neovascular AMD (nAMD) is characterized by choroidal neovascularization (CNV), vascular leakage, and fibrotic remodeling. Although anti-vascular endothelial growth factor (VEGF) therapy has improved visual outcomes for some patients, emerging findings highlight growing concerns regarding treatment resistance, persistent disease activity, and progression to subretinal fibrosis, which remain major challenges. Growing evidence indicates that nAMD arises from the interplay among metabolic dysfunction, oxidative stress, chronic inflammation, hypoxia signaling, and extracellular matrix remodeling within the outer retina. Disruption of mitochondrial function, lipid handling in the retinal pigment epithelium, complement activation, inflammasome signaling, macrophage immune-metabolic reprogramming, and hypoxia-driven VEGF expression collectively shape angiogenesis and lesion progression. Murine models, including laser-induced CNV, two-stage laser-induced fibrosis, and spontaneous or genetic systems such as JR5558, VLDLR-deficient, and CYP27A1-deficient mice, have provided essential mechanistic insights into these pathways. Aging and biological sex influence metabolism, mitochondrial efficiency, immune responses, hypoxia responsiveness, and wound-healing capacity. However, many widely used experimental models rely on young adult animals and often use a single sex without accounting for sex- or age-based differences, leaving these critical factors understudied and inadequately integrated into preclinical design. This review integrates current mechanistic understanding with a critical evaluation of murine nAMD models and emphasizes the importance of incorporating biological age and sex into experimental systems to improve mechanistic interpretation and translational relevance.

Keywords: Age-related macular degeneration, Retinal pigment epithelium, Retina, Inflammation, Immunosenescence, Choroidal neovascularization, Anti-VEGF resistance, Subretinal fibrosis, Murine models

1. Introduction

AMD is a leading cause of irreversible central vision loss among older adults and represents a growing clinical and socioeconomic burden as global life expectancy increases (Fleckenstein et al., 2021; Zhang et al., 2024). In the United States, nearly 20 million individuals are affected, with a substantial rise in prevalence among those aged 65 and older (Fleckenstein et al., 2024; Rein et al., 2022). AMD is broadly categorized into early and intermediate stages, characterized by drusen accumulation, retinal pigment epithelial (RPE) dysfunction, and late-stage disease, which later manifests as either geographic atrophy (GA) or nAMD (Ferris et al., 2013; Garcia-Layana et al., 2017; Danis et al., 2015; Pugazhendhi et al., 2021). Although the advanced stages contribute to visual impairment, nAMD accounts for the more rapid and profound central vision loss due to CNV, vascular leakage, and subsequent fibrotic remodeling (Shao et al., 2016; Little et al., 2018; Khanani et al., 2020).

Intravitreal anti-VEGF therapies, often administered in conjunction with nutritional supplementations based on the Age-Related Eye Disease Study (AREDS) formulations, have significantly improved outcomes in nAMD (Stanca et al., 2019; Ohr et al., 2012; Rezende et al., 2014; Age-Related Eye Disease Study Research and G., 2001). However, heterogeneous treatment responses, incomplete disease control, and progression to subretinal fibrosis remain common (Yang et al., 2016; Faatz et al., 2022; Daniel et al., 2014). These clinical challenges highlight the need for continued investigation into disease mechanisms and for the improved use of experimental models that accurately recapitulate disease progression and therapeutic resistance.

AMD risk and progression are influenced by both non-modifiable and modifiable factors, as shown in Fig. 1. Advanced age is the strongest risk factor, and epidemiological studies report sex-associated differences in prevalence and disease expression (Rudnicka et al., 2012). Additional risk factors include genetic predisposition, race, smoking, dyslipidemia, obesity, and cardiometabolic disease (Wong et al., 2014; Chakravarthy et al., 2010; Klein et al., 1993; Francis et al., 2007). Many of these risk factors involve shared biological pathways characterized by metabolic dysfunction, oxidative stress, complement activation, lipid dysregulation, and chronic inflammation (Ambati et al., 2013; Kauppinen et al., 2016; Curcio et al., 2009; Hollyfield et al., 2008; Rudnicka et al., 2015; Marin et al., 2022). Together, these processes contribute to immune and metabolic dysregulation in the aging retina and choroid, creating conditions that favor aberrant angiogenesis and fibrotic remodeling (Helotera et al., 2022; Ma et al., 2024; Yasuda et al., 2026; Dong et al., 2009; Nozaki et al., 2006; Wang et al., 2023).

Metabolic inflammation, characterized by chronic low-grade inflammation associated with obesity, dyslipidemia, and insulin resistance, predisposes individuals to type 2 diabetes mellitus, cardiovascular disease, and nonalcoholic steatohepatitis, all of which are associated with increased risk of AMD (Ghaem Maralani et al., 2015). Consistent with these findings, metabolomic profiling of nAMD has identified enrichment of metabolites associated with lipid and mitochondrial metabolism, including acylcarnitines involved in fatty acid β-oxidation, bile acids and their receptors, glycerophospholipids, and the amino acids taurine, citrulline, and arginine, with citrulline and arginine known to promote angiogenesis (Brown et al., 2018; Warden et al., 2025). Experimental studies support this association, as increased CNV severity and enhanced retinal microglial infiltration have been observed in mice receiving fecal microbiota from donors on a high-fat diet (Andriessen et al., 2016). Additional evidence of a metabolic-inflammatory axis within the retina includes the G-protein-coupled receptor GPR109A, a niacin-responsive receptor expressed in the RPE that regulates free fatty acid mobilization. Its expression increases under diabetic and fasting conditions, and pharmacologic activation reduces inflammatory markers in experimental models (Gambhir et al., 2012). These observations indicate that nAMD develops within a context of interacting metabolic and inflammatory disturbances rather than from a single molecular trigger. Aging is associated with declines in mitochondrial function and increased oxidative stress (Eells, 2019; Tong et al., 2022), shifts in macrophage phenotype and inflammatory signaling (Kelly et al., 2007; Rea et al., 2018), alterations in complement regulation (Gaya da Costa et al., 2018; Hoh Kam et al., 2013), and impaired tissue repair responses (Horrigan et al., 2026; Yi et al., 2023), each of which may contribute to increased susceptibility to pathologic angiogenesis (Espinosa-Heidmann et al., 2002). Biological sex is also associated with differences in immune activation, inflammatory signaling, complement activity, systemic and retinal lipid biology, and vascular regulation (Marin et al., 2022; Gaya da Costa et al., 2018; Klein et al., 2016; Fonteh et al., 2022; Colak et al., 2011; Slade et al., 2021; Schmidl et al., 2015; Stanhewicz et al., 2018). Age- and sex-related differences in immune and metabolic regulation therefore have the potential to influence CNV architecture, lesion progression, and fibrotic remodeling.

Importantly, these pathogenic mediators are not simply background features of disease but active determinants of the structural changes that characterize nAMD. Disruption of mitochondrial metabolism and lipid handling lowers the threshold for inflammatory activation, while complement dysregulation and macrophage reprogramming amplify immune signaling within the subretinal space (Nozaki et al., 2006; Feher et al., 2006; Markov et al., 2025; Sene et al., 2013; Combadiere et al., 2007). Hypoxia-induced VEGF expression drives endothelial proliferation and increases vascular permeability, while persistent inflammatory signaling, along with extracellular matrix remodeling, promotes lesion maturation and fibrovascular transformation (Ramakrishnan et al., 2014; Wang et al., 2017; Forsythe et al., 1996). In this way, the clinical manifestations of nAMD, including angiogenic complexity, lesion expansion, variable therapeutic responsiveness, and scar formation, reflect the downstream consequences of defined biological drivers operating within an aging retinal environment.

Murine models have played a central role in advancing understanding of how these mechanisms contribute to the different stages of AMD and in the development of novel therapies. Laser-induced CNV remains a widely used and reproducible murine model of pathological angiogenesis, supported by standardized protocols and optimization studies (Lambert et al., 2013; Gong et al., 2015). Additionally, spontaneous transgenic systems (e.g., JR5558, CYP27A1, and VLDLR-deficient mice) and fibrosis paradigms (e.g., two-stage laser models) have expanded the experimental repertoire (Nagai et al., 2014; Heckenlively et al., 2003; Saadane et al., 2014; Little et al., 2020). These systems enable the study of distinct stages of AMD in isolation and, when combined with genetic mutations, allow interrogation of the multifactorial retinal environment. They have been refined for lesion induction, angiogenesis, and therapeutic evaluation. However, many commonly used models rely on young adult animals and frequently use a single sex, without accounting for sex-specific differences. Given that nAMD is an age-associated disease with documented sex-related differences in immune and metabolic regulation, limited incorporation of biological age and sex into experimental design may affect interpretation and translational applicability. Previous reviews have emphasized that while AMD experimental models are invaluable, no single system fully captures the complexity of human disease. This highlights the need for continued refinement of model systems, along with a critical understanding of their limitations and careful consideration of whether they appropriately address the research question (Pennesi et al., 2012; Rastoin et al., 2020).

In this review, we evaluate commonly used murine models of nAMD, considering age- and sex-associated differences in immune-metabolic regulation. Our aim is to examine how these biological factors may influence disease mechanisms, model behavior, and therapeutic responsiveness, and to highlight the importance of incorporating biological context into preclinical study design.

2. Types of age-related macular degeneration: wet and dry

2.1. Dry (atrophic) age-related macular degeneration

AMD is a multifocal retinal disease broadly categorized into two clinical forms: dry (atrophic) and wet (neovascular) AMD, with progressive transition from early and intermediate to advanced disease stages as shown in Fig. 2. Dry AMD accounts for approximately 90% of all AMD cases and is primarily associated with lipid accumulation above Bruch’s membrane, which further contributes to RPE dysfunction. In the healthy retina, Bruch’s membrane forms a thin, semipermeable barrier between the choriocapillaris and the RPE, facilitating metabolic exchange and maintaining retinal homeostasis (Bhutto et al., 2012). The RPE lies adjacent to the photoreceptor outer segments and plays a critical role in photoreceptor maintenance through daily phagocytosis of shed outer segment discs, regulation of oxidative stress, and transport of nutrients and waste products.

Fig. 2. — Clinical progression of age-related macular degeneration.

AMD progresses from early and intermediate stages, characterized by drusen accumulation and RPE pigmentary changes, to advanced disease. Geographic atrophy (dry AMD) involves degeneration of the RPE and photoreceptors, whereas neovascular (wet) AMD is defined by CNV, in which abnormal vessels breach Bruch’s membrane and invade the subretinal space. CNV leads to vascular leakage, hemorrhage, and ultimately subretinal fibrosis and fibrotic scar formation, major drivers of rapid and irreversible vision loss in nAMD. The illustration was created using BioRender. AMD, age-related macular degeneration; RPE, retinal pigment epithelium; CNV, choroidal neovascularization; nAMD, neovascular age-related macular degeneration.

As the retina ages and is exposed to oxidative stress, RPE function becomes compromised, leading to impaired clearance of metabolic byproducts and disruption of Bruch’s membrane integrity. These changes promote the accumulation of extracellular, lipid- and protein-rich deposits known as drusen between the RPE and Bruch’s membrane, as illustrated in Fig. 2 (Handa et al., 2019; Rabin et al., 2013). Progressive drusen deposition and RPE dysfunction contribute to structural and functional retinal decline and can ultimately lead to GA, the advanced stage of dry AMD, characterized by irreversible loss of the RPE, photoreceptors, and the underlying choriocapillaris. Clinically, fundus imaging reveals areas of macular depigmentation corresponding to RPE and photoreceptor loss, while optical coherence tomography (OCT) provides cross-sectional visualization of retinal thinning, RPE atrophy, and drusen accumulation associated with advanced dry AMD (Ly et al., 2017; Yang et al., 2021).

Despite extensive characterization of dry AMD pathology, therapeutic options remain limited. Previous studies proposed that reducing drusen burden might slow disease progression; however, evidence suggests that drusen represents a downstream consequence rather than a primary driver of the disease. As a result, recent therapeutic development has focused primarily on GA. Pegcetacoplan, a complement inhibitor targeting C3 and C3b, has demonstrated approximately a 27% reduction in GA lesion growth at 12 months in clinical trials (Heier et al., 2023). Nonetheless, treatment efficacy remains influenced by dosing frequency, patient compliance, and genetic background, underscoring the continued need for more effective disease-modifying therapies.

2.2. Wet (neovascular) age-related macular degeneration

In contrast, nAMD is less prevalent, accounting for approximately 10% of AMD cases, yet it is responsible for the majority of AMD-associated vision loss. Age-related thickening and calcification of Bruch’s membrane impairs oxygen and nutrient diffusion from the choriocapillaris to the outer retina, exacerbating hypoxia and chronic RPE stress (Hussain et al., 2010; McHugh et al., 2019; Kurihara et al., 2016). These conditions promote the transition to exudative nAMD, in which immature blood vessels develop beneath and through the RPE, as shown in Fig. 2. This pathological neovascular growth penetrates Bruch’s membrane and the RPE, a process referred to as CNV.

CNV is the defining pathological feature of nAMD and frequently arises during progression from intermediate or advanced dry AMD (Douglas et al., 2022; Wakatsuki et al., 2023). Patients with nAMD may experience central scotomas similar to those observed in dry AMD; however, nAMD is often distinguished by acute or rapidly progressive vision loss (Stahl, 2020). A common and severe consequence of CNV is subretinal fibrosis, which results from aberrant wound-healing responses and leads to permanent damage to photoreceptors, the RPE, and the choriocapillaris (Ishikawa et al., 2016; Tenbrock et al., 2022). Time-course studies indicate that subretinal fibrosis develops in approximately 45% of eyes within 2 years of anti-VEGF therapy and persists in over 40% of eyes at 10 years post-treatment (Tenbrock et al., 2022; Liu et al., 2024). Eyes with subfoveal fibrotic scarring following anti-VEGF therapy exhibit the poorest visual prognosis.

Diagnosis of nAMD relies on multimodal imaging, including fluorescein angiography and OCT, to confirm the presence and activity of CNV. Although anti-VEGF therapies effectively suppress neovascular activity and reduce the rate of angiogenesis (Nagai et al., 2014), they require repeated intravitreal administration and remain invasive. Clinical evidence indicates that anti-VEGF treatment does not eliminate the risk of fibrotic remodeling and may, in some cases, be associated with progression of subretinal scarring (Ishikawa et al., 2016). These limitations highlight the need for therapeutic approaches that address not only angiogenesis but also the downstream fibrotic and degenerative processes that contribute to irreversible vision loss in nAMD.

3. Cellular and molecular mechanisms in AMD pathogenesis

3.1. Metabolic dysregulation

AMD is increasingly recognized as a disease rooted in metabolic failure within the outer retinal environment (Ferrington et al., 2017; Riazi-Esfahani et al., 2017). The outer retina is among the most metabolically active tissues in the body (Country, 2017; Wong-Riley, 2010), requiring precise coordination of energy production, substrate utilization, and lipid metabolism to sustain photoreceptor function and tissue integrity. Even subtle disruptions to this metabolic balance can impose chronic stress on AMD-vulnerable tissues (Ferrington et al., 2017; Riazi-Esfahani et al., 2017). As illustrated in Fig. 3, metabolic dysregulation in the outer retina promotes mitochondrial dysfunction and accumulation of reactive oxygen species, establishing a key upstream driver of oxidative stress in nAMD (Ferrington et al., 2017; Kanow et al., 2017; Murphy, 2009).

Fig. 3. — Cellular and molecular mechanisms in nAMD pathogenesis.

Early pathogenic events in neovascular age-related macular degeneration (nAMD) are driven by metabolic dysfunction, oxidative stress, chronic inflammation, and age-associated immunosenescence within the retinal pigment epithelium (RPE)–choroidal complex. Mitochondrial dysfunction promotes the accumulation of reactive oxygen species (ROS), which activate inflammatory signaling and stabilize hypoxia-inducible factor-1 alpha (HIF-1α) under metabolically stressed conditions. HIF-1α–mediated hypoxic signaling upregulates vascular endothelial growth factor (VEGF), thereby promoting VEGF-VEGFR2–dependent pathological angiogenesis and the formation of choroidal neovascularization (CNV) lesions characteristic of nAMD. Following CNV formation, persistent inflammatory signaling further contributes to lesion progression and drives profibrotic remodeling by activating transforming growth factor-beta (TGF-β) signaling pathways. TGF-β–mediated extracellular matrix (ECM) remodeling, characterized by increased matrix metalloproteinase (MMP) activity and collagen deposition, promotes epithelial-to-mesenchymal transition (EMT) in RPE cells and endothelial-to-mesenchymal transition (EndoMT) in CNV-associated endothelial cells. These mesenchymal transitions facilitate myofibroblast differentiation within the fibrovascular membrane, ultimately leading to excessive ECM accumulation and the development of subretinal fibrosis. Collectively, this cascade links early immune–metabolic dysfunction to angiogenesis-driven CNV and the subsequent fibrotic scarring observed in advanced stages of nAMD. The illustration was created using BioRender. nAMD, neovascular age-related macular degeneration; RPE, retinal pigment epithelium; ROS, reactive oxygen species; HIF-1α, hypoxia-inducible factor-1 alpha; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2; CNV, choroidal neovascularization; TGF-β, transforming growth factor-beta; ECM, extracellular matrix; MMP, matrix metalloproteinase; EMT, epithelial-to-mesenchymal transition; EndoMT, endothelial-to-mesenchymal transition.

At the center of this metabolic vulnerability lies the RPE, which operates under high energetic demand. In addition to supporting photoreceptor survival, the RPE must continuously process large quantities of lipid-rich material while maintaining intracellular energy balance. Experimental studies demonstrate that the RPE is metabolically specialized, relying heavily on mitochondrial oxidative metabolism and fatty acid β-oxidation to meet its energy requirements and support the metabolic needs of the adjacent neural retina (Kanow et al., 2017; Adijanto et al., 2014a, 2014b). During aging, mitochondrial efficiency declines, reducing metabolic flexibility and limiting the ability to adapt to increased energetic or substrate demands (Feher et al., 2006; Karunadharma et al., 2010).

Age-associated changes at the level of Bruch’s membrane and the choroidal vasculature further exacerbate metabolic strain. Progressive lipid accumulation and structural remodeling impair nutrient diffusion and waste clearance, while reduced choroidal perfusion restricts oxygen delivery. Together, these changes impose sustained metabolic pressure on the outer retina, requiring compensatory adjustments that may become maladaptive over time. Importantly, such metabolic stress can arise well before overt structural degeneration is clinically detectable (Biesemeier et al., 2014; McLeod et al., 2009).

Systemic metabolic disorders amplify these local vulnerabilities. Epidemiological studies have consistently demonstrated associations between AMD progression and metabolic syndrome components such as obesity, dyslipidemia, and insulin resistance, highlighting a potential role for systemic metabolic dysfunction in modulating retinal disease risk and severity (Ghaem Maralani et al., 2015; Seddon et al., 2003). One common metabolic disorder is diabetes mellitus, which leads to a constellation of metabolic disturbances affecting retinal energy utilization, mitochondrial efficiency, and vascular support. Chronic hyperglycemia alters substrate availability and impairs mitochondrial respiration, while diabetes-associated microvascular dysfunction compromises choroidal nutrient and oxygen delivery. These systemic perturbations increase metabolic demand within the outer retina and accelerate the transition from metabolic compensation to metabolic failure. Translational support for the role of metabolic dysregulation in AMD comes from metabolomic profiling studies, which have identified alterations in circulating metabolites associated with mitochondrial energy production and lipid metabolism in patients with nAMD (Lains et al., 2019; Hou et al., 2020). Enrichment of metabolites associated with fatty acid oxidation, including acylcarnitines and components of the carnitine shuttle pathway, suggests impaired mitochondrial fuel handling in advanced disease. While these observations are associative, they align with experimental evidence identifying mitochondrial lipid metabolism as a critical vulnerability in AMD-relevant tissues. The magnitude of metabolic dysregulation is not uniform across biological contexts. Aging further modifies metabolic resilience within the outer retina. Declines in mitochondrial biogenesis, reduced efficiency of oxidative phosphorylation, and increased susceptibility to reactive oxygen species collectively narrow the adaptive range of the RPE (Eells, 2019; Feher et al., 2006; Jarrett et al., 2012). Age-associated shifts in macrophage metabolism toward a more glycolytic, pro-inflammatory phenotype may also influence local lipid handling and inflammatory tone within the subretinal space (Kelly et al., 2007; Espinosa-Heidmann et al., 2002). Concurrently, impaired mitochondrial lipid oxidation and reduced metabolic flexibility may amplify inflammatory activation, lowering the threshold for angiogenic and fibrotic responses (Ma et al., 2024; Eells, 2019; Kelly et al., 2007; Jarrett et al., 2012; Joyal et al., 2016). Biological sex introduces an additional layer of metabolic modulation. Sex hormones influence mitochondrial dynamics and lipid metabolism in multiple tissues, including the retina (Giddabasappa et al., 2010; Elliot et al., 2010). Estrogen signaling has been associated with enhanced mitochondrial efficiency and antioxidant capacity, whereas hormonal decline may alter lipid handling and increase oxidative burden (Simpkins et al., 2010; Razmara et al., 2007; Zhao et al., 2021; Cousins et al., 2003). Differences in systemic lipid profiles, fatty-acid utilization, and immune activation between males and females may therefore influence the metabolic state of AMD-vulnerable tissues. Experimental models demonstrate sex-dependent differences in inflammatory and angiogenic responses (Espinosa-Heidmann et al., 2005), suggesting that metabolic-immune interactions are not identical across sexes.

3.2. Oxidative stress

As previously noted, the retina is inherently susceptible to oxidative stress due to its high metabolic activity. Consequently, it has elevated oxygen consumption, membranes enriched in polyunsaturated fatty acids, and continuous exposure to visible light (Country, 2017; Wong-Riley, 2010; Ames et al., 1993). Essential for visual function, these features nevertheless make the outer retina particularly vulnerable to the progressive accumulation of reactive oxygen species (ROS) over time (Country, 2017; Beatty et al., 2000). With aging, the capacity of endogenous antioxidant systems declines, allowing oxidative stress to emerge as a major contributor to retinal dysfunction in AMD (Jarrett et al., 2012; Kauppinen et al., 2012).

Mitochondria are a dominant endogenous source of ROS, particularly under conditions of metabolic strain and impaired electron transport (Ferrington et al., 2017; Murphy, 2009). Age-associated declines in mitochondrial efficiency and quality control increase ROS generation in retinal cells, including the RPE (Ferrington et al., 2017; Karunadharma et al., 2010). In parallel, photooxidative mechanisms become increas ingly relevant as photoreactive byproducts accumulate within the aging RPE (Wielgus et al., 2010; Davies et al., 2001). Together, mitochondrial and light-dependent ROS production establishes a chronic oxidative environment that challenges retinal homeostasis. As illustrated in Fig. 3, ROS accumulation provides a mechanistic bridge between oxidative injury and downstream inflammatory, hypoxia-associated, and VEGF-dependent pathways that contribute to CNV formation and fibrotic remodeling in nAMD (Ferrington et al., 2017; Murphy, 2009; Datta et al., 2017; Inoue et al., 2007; Kwak et al., 2000). A prominent manifestation of cumulative oxidative stress in the RPE is the progressive accumulation of lipofuscin, an auto fluorescent material derived from incomplete degradation of photoreceptor outer segments (Eldred et al., 1988; Boyer et al., 2012). While lipofuscin is a feature of normal aging, excessive accumulation is strongly associated with AMD. Lipofuscin granules are primarily localized to RPE lysosomes and often form complexes with melanin, referred to as melanolipofuscin (Feeney, 1978). Among the best-characterized lipofuscin components is the bisretinoid A2E, which is generated through reactions involving retinaldehyde and phosphatidylethanolamine (Sparrow et al., 2005; Ben-Shabat et al., 2002). A2E is highly photoreactive and, upon light exposure, promotes ROS formation and phototoxic injury to RPE cells.

As lipofuscin and melanolipofuscin occupy an increasing fraction of the RPE cytoplasm with age, the photoreceptor-RPE interface becomes increasingly susceptible to photooxidative damage. Oxidative modifications of lipids, proteins, and nucleic acids accumulate, gradually eroding cellular resilience and functional reserve (Jarrett et al., 2012; Beatty et al., 2000). Importantly, oxidative stress in the aging retina does not remain confined to molecular damage alone. Instead, it also serves as a critical upstream signal that alters cellular behavior and primes the tissue microenvironment for subsequent inflammatory activation, providing a mechanistic link to the chronic inflammatory processes observed in AMD (Hollyfield et al., 2008; Kauppinen et al., 2012). The magnitude and consequences of oxidative stress are further influenced by biological context. Aging not only increases cumulative oxidative burden but also alters the efficiency of antioxidant defenses and cellular repair mechanisms. Declines in glutathione availability, reduced activity of key antioxidant enzymes, particularly catalase, have been reported with aging and are consistent with diminished capacity for ROS detoxification in older tissues (Samiec et al., 1998; Frank et al., 1999; Miyamura et al., 2004; Liles et al., 1991). Age-associated mitochondrial DNA (mtDNA) damage and reduced mtDNA repair further perpetuate oxidative injury and correlate with AMD severity (Karunadharma et al., 2010; Lin et al., 2011). Biological sex may also modulate oxidative stress responses. In RPE models, 17β-estradiol protects against oxidative stress via ERβ-dependent mechanisms, including preservation of mitochondrial function, reduced ROS production, and induction of antioxidant genes (Giddabasappa et al., 2010; Wei et al., 2018). Estrogen has also shown protective effects in oxidative injury paradigms relevant to retinal degeneration in vivo and in human RPE injury models (Wei et al., 2018). Differences in systemic lipid profiles and oxidative susceptibility have been reported in AMD cohorts in a sex-associated manner, and sex-stratified analyses of systemic factors further support biologically meaningful heterogeneity between males and females (Colak et al., 2011; Sasaki et al., 2018; Li et al., 2024). Variability in hormonal status, particularly with advancing age, may therefore contribute to differences in oxidative resilience across sexes.

3.3. Inflammation

Inflammation in AMD is increasingly recognized as a chronic, maladaptive response to persistent tissue stress rather than an acute reaction to injury (Kauppinen et al., 2016; Wong et al., 2022; Xu et al., 2009). The RPE plays a central role in shaping this inflammatory milieu. In addition to its supportive and metabolic functions, the RPE acts as an immunologically active tissue that senses cellular stress and produces cytokines, chemokines, and other immune mediators that influence local and systemic immune responses (Datta et al., 2017; Xu et al., 2009; Klettner et al., 2021). With aging and sustained stress exposure, these protective responses can become dysregulated, contributing to chronic inflammation within the outer retina.

One major pathway linking cellular stress to inflammation in AMD is activation of redox-sensitive transcription factors, including nuclear factor-κB (NF-κB). Oxidative stress, lysosomal dysfunction, and mitochondrial damage promote NF-κB activation in RPE cells, leading to increased expression of proinflammatory cytokines, chemokines, and angiogenic mediators such as VEGF (Datta et al., 2017; Feng et al., 2019; Marazita et al., 2016; Chen et al., 2015). Sustained activation of these pathways disrupts RPE barrier integrity and alters paracrine signaling among the RPE, photoreceptors, and choroidal endothelium (Datta et al., 2017; Wong et al., 2022; Marazita et al., 2016). Innate immune activation through inflammasome signaling represents another well-characterized inflammatory mechanism in AMD. The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is activated in response to intracellular stress signals such as lysosomal membrane permeabilization, mitochondrial dysfunction, and accumulation of oxidized lipids (Kauppinen et al., 2016; Anderson et al., 2013; Brandstetter et al., 2015a, 2015b). Experimental studies demonstrate that destabilization of RPE lysosomes, including those induced by lipofuscin-associated bisretinoid components such as N-retinylidene-N-retinylethanolamine (A2E), can activate the NLRP3 inflammasome and promote caspase-1-dependent processing and release of interleukin-1 (IL-1) family cytokines (Anderson et al., 2013; Brandstetter et al., 2015a, 2015b). These data provide a direct mechanistic link between age-related waste accumulation, oxidative stress, and innate immune activation in the retina. Inflammation in AMD is not confined to cell-intrinsic signaling within the RPE; it is amplified by extracellular deposits and humoral immunity. Drusen and basal deposits accumulate between the RPE and Bruch’s membrane and act as chronic inflammatory scaffolds enriched in oxidized lipids and proteins, including complement components and complement activation products (Nozaki et al., 2006; Crabb, 2014; Johnson et al., 2001, 2011). Proteomic and histologic studies have identified complement constituents within drusen and along the RPE-Bruch’s membrane interface, supporting the concept that dysregulated complement activity sustains low-grade inflammatory injury and promotes downstream immune cell recruitment (Nozaki et al., 2006; Johnson et al., 2001, 2011).

Complement-driven inflammation couples closely to cellular immunity in the aging retina. Activated microglia and infiltrating macrophages accumulate in the subretinal space in AMD and AMD-like states, where they can exacerbate tissue injury through the secretion of cytokines, reactive oxygen species, proteases, and proangiogenic mediators (Kauppinen et al., 2016; Wong et al., 2022; Borchert et al., 2023; Szczepan et al., 2025). Seminal work has linked chemokine signaling (e.g., CX3CR1 pathways) to subretinal microglial accumulation and AMD-like features, underscoring that immune cell trafficking and persistence are central determinants of disease chronicity (Combadiere et al., 2007; Tuo et al., 2007; Luhmann et al., 2013).

As inflammatory signaling persists, it contributes to progressive disruption of RPE barrier function, altered communication between retinal and choroidal compartments, and changes in the expression of factors that regulate vascular and tissue remodeling. Over time, this sustained inflammatory environment amplifies tissue vulnerability and reinforces degenerative processes within the outer retina (Kauppinen et al., 2016; Wong et al., 2022; Mullins et al., 2014). In this framework, inflammation emerges not as an isolated pathogenic event, but as a downstream consequence of cumulative metabolic and oxidative stress that perpetuates AMD progression. As illustrated in Fig. 3, inflammatory signaling links upstream metabolic and oxidative stress to NF-κB–dependent cytokine production, VEGF activation, and TGF-β–associated remodeling, thereby linking chronic immune activation to CNV progression and subretinal fibrosis in nAMD (Kauppinen et al., 2016; Datta et al., 2017; Krause et al., 2014; Yeo et al., 2019; Zhang et al., 2012). The magnitude and persistence of inflammatory activation are strongly influenced by biological context and are directly relevant to neovascular AMD. Aging alters innate immune regulation in the retina and choroid, shifting the immune tone toward a sustained pro-inflammatory state characterized by microglial activation and altered macrophage function (Chen et al., 2019; Ma et al., 2016). In experimental CNV models, age-associated changes in macrophage phenotype are accompanied by altered cytokine production and reduced anti-angiogenic regulation, thereby contributing to increased lesion severity (Kelly et al., 2007). These immune alterations may prolong complement activation and inflammasome signaling within the subretinal space, processes that are mechanistically linked to angiogenic amplification and fibrotic remodeling (Doyle et al., 2012; Faber et al., 2012).

Age-related increases in complement component expression and deposition in the RPE/choroid complex have been documented in both aging tissue and AMD retina (Marin et al., 2022; Hoh Kam et al., 2013; Faber et al., 2012). Furthermore, age-related impairments in autophagic and lysosomal function in the RPE may sustain inflammasome activation and interleukin release, thereby reinforcing pro-angiogenic and pro-fibrotic signaling cascades (Kaarniranta et al., 2013; Cox et al., 2025). Biological sex may further modulate inflammatory pathways relevant to nAMD progression. Sex-dependent differences in immune activation and complement factor profiles have been reported in AMD cohorts (Marin et al., 2022; Klein et al., 2016), and estrogen signaling has been shown to regulate NF-κB activity and cytokine production in RPE cells (Paimela et al., 2007). Given the established role of inflammatory cytokines and complement activation in CNV development, sex-dependent variation in immune tone may contribute to differences in angiogenic architecture, lesion persistence, and fibrotic outcomes in experimental and clinical nAMD.

3.4. Immunosenescence

The shift from acute, protective inflammation to chronic, maladaptive inflammation in AMD is strongly shaped by immunosenescence. This refers to the progressive, age-related decline in immune function, a recognized hallmark of aging (Goyani et al., 2024). Aging is accompanied by thymic involution and reduced naïve T-cell output, which alter immune surveillance and weaken mechanisms that normally constrain tissue inflammation (Liang et al., 2022; Thomas et al., 2020). This immune remodeling promotes inflammaging, a state of persistent low-grade inflammatory signaling that lowers the threshold for retinal injury and impairs the restoration of immune quiescence (Fulop et al., 2023). In this context, drusen-associated lipids can acquire immunogenic properties; for example, docosahexaenoic acid (DHA)-derived oxidative adducts, such as carboxyethylpyrrole (CEP), can act as neoantigens, and human studies have detected CEP adducts and anti-CEP autoantibodies at higher levels in AMD than in controls (Hollyfield et al., 2008; Gu et al., 2003). Experimental work further supports the pathogenic potential: immunization with CEP-adducted proteins induces AMD-like retinal lesions in mice, providing a mechanistic link among oxidative lipid damage, loss of tolerance, and immune-mediated retinal injury (Hollyfield et al., 2008).

In addition to adaptive immune dysfunction, immunosenescence affects resident and infiltrating innate immune cells within the retina. Under physiological conditions, macrophages contribute to retinal homeostasis by clearing cellular debris and maintaining immune quiescence. With aging, macrophage function becomes impaired, reducing clearance capacity and facilitating drusen accumulation (Apte et al., 2006; Hageman et al., 2001). Microglia, the resident immune cells of the retina, similarly exhibit age-related changes characterized by chronic activation, impaired resolution of inflammation, and adoption of an amoeboid morphology. These alterations promote sustained proinflammatory signaling and are consistently associated with AMD pathology (Damani et al., 2011; Alves et al., 2020).

Cellular senescence further intersects with immune dysfunction in AMD. Senescence refers to a stable state of cell-cycle arrest that occurs in response to accumulated cellular damage (Campisi et al., 2007). Aging tissues exhibit increased susceptibility to DNA damage, oxidative stress, and accumulation of toxic byproducts, including drusen-associated components such as amyloid-beta peptides and A2E, which are derived from photoreceptor degeneration (Hollyfield et al., 2008; Sparrow et al., 2005; Johnson et al., 2002). Senescent cells adopt a senescence-associated secretory phenotype (SASP), characterized by the release of proinflammatory cytokines, growth factors, and matrix-remodeling enzymes, including IL-1β, IL-6, IL-8, VEGF, and matrix metalloproteinase 9 (MMP9), along with impaired autophagy and reduced complement factor H activity (Kaarniranta et al., 2013; Coppe et al., 2010; Chae et al., 2021). These changes promote extracellular matrix deposition, increased vascular permeability, and disruption of the blood-retina barrier. Together, immunosenescence and senescence provide a mechanistic explanation for why RPE stress signaling and innate immune activation in AMD frequently fail to resolve and instead evolve into a self-sustaining inflammatory state that promotes barrier dysfunction, angiogenic remodeling, and fibrotic progression.

As illustrated in Fig. 3, immunosenescence amplifies the transition from unresolved inflammatory signaling to VEGF-associated neovascular remodeling and TGF-β–linked fibrotic progression, thereby connecting age-dependent immune dysfunction with late nAMD pathology (Kauppinen et al., 2016; Hollyfield et al., 2008; Thomas et al., 2020; Alves et al., 2020). Biological sex influences multiple dimensions of immune aging. Females generally exhibit more robust innate and adaptive immune responses across the lifespan but may also experience greater inflammatory persistence under chronic stress conditions (Klein et al., 2016; Calabro et al., 2023; Yanicke et al., 2023). Sex hormones modulate macrophage differentiation, cytokine production, and complement activity, and hormonal changes with aging may alter immune regulation. Differences in immune cell composition, activation thresholds, and inflammatory resolution between males and females may therefore contribute to variability in lesion severity and progression.

3.5. Hypoxia signaling

The outer retina has one of the highest metabolic demands in the body and relies on continuous oxygen delivery from the choriocapillaris. With aging, structural and vascular changes at the level of the RPE-Bruch’s membrane-choroid complex can limit oxygen diffusion. Age-associated reductions in choroidal blood flow and attenuation of the choriocapillaris have been documented and may contribute to localized hypoxic stress in the macula (Grunwald et al., 1998; Emeterio Nateras et al., 2014; Boltz et al., 2010). In this setting, reduced oxygen availability can alter RPE metabolism, immune signaling, and vascular homeostasis. Cellular adaptation to hypoxia is mediated primarily through stabilization of hypoxia-inducible factors (HIFs), particularly HIF-1α. Under normoxic conditions, HIF-1α undergoes rapid proteasomal degradation. Hypoxia inhibits this degradation, allowing HIF-1α to accumulate, translocate to the nucleus, and activate transcription of genes involved in angiogenesis, inflammation, glycolysis, and cell survival. Expression of HIF-1α and HIF-2α has been detected in human choroidal neovascular membranes associated with AMD, supporting activation of hypoxia-responsive pathways within pathologic lesions (Inoue et al., 2007). Experimental studies further demonstrate that RPE-derived HIF-1α contributes to lesion development in laser-induced CNV models, with genetic suppression of HIF signaling resulting in reduced CNV area and decreased expression of downstream angiogenic mediators (Lin et al., 2012). Hypoxia-induced HIF activation promotes VEGF transcription, a central driver of pathologic angiogenesis. As illustrated in Fig. 3, hypoxia-driven stabilization of HIF-1α links metabolic and oxidative stress to VEGF–VEGFR2 signaling, thereby promoting CNV formation and contributing to downstream fibrovas cular remodeling in nAMD (Xie et al., 2021). In addition to VEGF, HIF-1α regulates the expression of adhesion molecules and chemokines that facilitate endothelial activation and leukocyte recruitment. Hypoxia, therefore, functions upstream of multiple pro-angiogenic and pro-inflammatory signals that converge on CNV formation. The magnitude and duration of HIF activation influence endothelial proliferation, vascular permeability, and lesion architecture. Hypoxia also interacts with inflammatory signaling pathways within the RPE and surrounding tissues. In vitro studies demonstrate that low oxygen tension can enhance the expression of inflammasome components and increase the production of pro-inflammatory cytokines in RPE cells (Doktor et al., 2018). Crosstalk between HIF-1α signaling and NF-κB pathways has been reported, suggesting that hypoxic stress may amplify inflammatory responses rather than acting solely through angiogenic mediators (Lu et al., 2014). These interactions provide a mechanistic link between oxygen deprivation, innate immune activation, and sustained lesion progression. In addition to angiogenesis and inflammation, hypoxia influences tissue remodeling. Reduced oxygen availability alters cellular metabolism, promotes epithelial-mesenchymal transition-associated changes in RPE cells, and can enhance profibrotic signaling in the presence of transforming growth factor (TGF-β) (Suzuki et al., 2022). HIF-1α has been implicated in regulating extracellular matrix components and fibrotic pathways in ocular tissues (Xie et al., 2021), suggesting that hypoxia contributes to the transition from active neovascularization to fibrovascular scarring.

Biological context modifies hypoxia signaling. Aging is associated with reduced vascular reserve, altered mitochondrial efficiency, and increased susceptibility to oxidative and metabolic stress, factors that may influence the threshold and consequences of HIF activation. Biological sex can also affect hypoxia-responsive pathways. Estrogen signaling has been shown to modulate HIF-1α and VEGF induction under varying oxygen conditions, and sex-associated differences in vascular regulation may alter tissue oxygen dynamics (Miyamoto et al., 2002; Kazi et al., 2007). Variability in oxygen delivery, endocrine signaling, and immune responsiveness, therefore, has the potential to influence the intensity and downstream effects of hypoxia-driven angiogenic and inflammatory programs.

3.6. VEGF signaling

VEGF is a central regulator of pathologic angiogenesis in neovascular AMD. VEGF-A is produced by multiple retinal and choroidal cell types, including RPE cells, Müller glia, macrophages, and endothelial cells (Krause et al., 2014; Le et al., 2010; Saint-Geniez et al., 2008). Under physiologic conditions, VEGF supports the maintenance of the choriocapillaris and vascular homeostasis. In nAMD, dysregulated VEGF expression promotes endothelial proliferation, migration, and survival, and increases vascular permeability, contributing to CNV and exudation (Kwak et al., 2000). VEGF-A exerts many of its endothelial effects through VEGF receptor-2 (VEGFR2), a receptor tyrosine kinase that activates downstream signaling programs governing survival, proliferation, and promotes permeability through Src-linked junctional regulation (Claesson-Welsh, 2016; Wang et al., 2020). Persistent VEGFR2 activation contributes to aberrant vessel growth, disorganized vascular architecture, and leakage characteristic of nAMD lesions. Genetic and pharmacologic inhibition of VEGFR2 similarly attenuates CNV formation (Kwak et al., 2000; Gu et al., 2010). These findings form the basis for current clinically used anti-VEGF therapies, including ranibizumab and aflibercept, which target VEGF-A or its receptor interactions to suppress neovascular growth and exudation (Rosenfeld et al., 2006; Heier et al., 2012). Despite therapeutic VEGF suppression, lesion recurrence and progression to fibrovascular scarring remain common in a subset of patients, suggesting that VEGF signaling operates within a broader network of inflammatory, metabolic, and remodeling pathways rather than acting as an isolated driver (Rofagha et al., 2013; Fu et al., 2024). Inflammatory cytokines, complement activation products, and hypoxia-associated signals can augment VEGF expression, thereby reinforcing the angiogenic drive (Nozaki et al., 2006; Nagineni et al., 2012). Fig. 3 highlights VEGF–VEGFR2 signaling as a central downstream convergence point through which metabolic stress, oxidative injury, inflammation, and hypoxia drive pathological angiogenesis and CNV formation in nAMD. In addition, macrophages recruited to CNV lesions serve as a source of VEGF and other pro-angiogenic mediators, linking innate immune activation to endothelial proliferation (Krause et al., 2014).

Aging influences VEGF signaling dynamics through multiple mechanisms. Age-associated alterations in mitochondrial function, oxidative stress, and immune tone can increase basal inflammatory signaling and modify endothelial responsiveness to angiogenic stimuli. Structural changes in the choriocapillaris and Bruch’s membrane may further alter VEGF gradients and receptor activation patterns within the subretinal space. Experimental studies demonstrate that aged mice develop larger and more complex CNV lesions than young adults, consistent with altered angiogenic regulation in the aging microenvironment (Espinosa-Heidmann et al., 2002). Biological sex may also modulate VEGF pathway activity. Experimental studies demonstrate sex- and estrogen-associated differences in CNV severity, supporting endocrine modulation of angiogenic and inflammatory responses in CNV lesions (Espinosa-Heidmann et al., 2005).

3.7. Extracellular matrix remodeling

ECM remodeling is a defining feature of lesion progression in neovascular AMD. The development of CNV requires the degradation of Bruch’s membrane and the restructuring of the subretinal extracellular environment to permit endothelial invasion (Yeo et al., 2019; Grossniklaus et al., 2010). MMPs, particularly MMP-2 and MMP-9, are upregulated in CNV lesions and facilitate extracellular matrix breakdown, endothelial migration, and neo-vascular extension. Experimental inhibition or genetic modulation of MMP activity has been shown to alter CNV size and vascular complexity in murine models, underscoring the importance of matrix remodeling in angiogenic expansion (Lambert et al., 2002, 2003). Beyond angiogenesis, ECM remodeling contributes to the transition from active neovascularization to fibrovascular scarring. Fig. 3 positions ECM remodeling as a downstream effector of inflammatory, hypoxic, and TGF-β–associated signaling, linking CNV progression to subretinal fibrosis in nAMD. In murine models of subretinal fibrosis secondary to CNV, extracellular matrix components such as collagen and fibronectin accumulate within subretinal lesions alongside neovascular tissue (Little et al., 2020).

TGF-β signaling plays a central role in this process, promoting myofibroblast differentiation and extracellular matrix deposition (Zhang et al., 2012; Zhao et al., 2022). Persistent remodeling alters lesion architecture, increases tissue stiffness, and contributes to irreversible visual decline (Little et al., 2018). Aging modifies extracellular matrix composition and turnover. Age-related structural changes in human Bruch’s membrane include thickening and alterations of the elastic layer, with measurable declines in elasticity/permeability that may affect transport properties and local tissue mechanics (Huang et al., 2007; Ugarte et al., 2006) (Little et al., 2018; Khanani et al., 2020; Stanca et al., 2019). These age-dependent changes can influence endothelial invasion dynamics and growth-factor distribution across the RPE-Bruch’s membrane-choroid interface (Huang et al., 2007; Ugarte et al., 2006).

Biological sex may also influence matrix remodeling in AMD-relevant contexts. Experimental studies show that sex and estrogen status modify CNV severity in laser-induced models (Espinosa-Heidmann et al., 2005), and in RPE systems, female sex hormones have been reported to suppress TGF-β2-induced epithelial-mesenchymal transition (EMT) programs linked to fibrotic remodeling (Giddabasappa et al., 2010; Kimura et al., 2014).

4. Experimental murine models for studying nAMD

4.1. Single-hit laser-induced CNV in mice

CNV is a defining pathological feature of neovascular AMD and is characterized by the growth of disorganized blood vessels from the choroid through Bruch’s membrane into the avascular subretinal space (Grossniklaus et al., 2010). This aberrant vascular expansion disrupts retinal architecture, compromises photoreceptor organization, and exacerbates oxidative and inflammatory stress within the outer retina (Kauppinen et al., 2016; Ambati et al., 2012). To model this process experimentally, as illustrated in Fig. 4, CNV is commonly induced in vivo in pigmented C57BL/6J mice by single-hit laser photocoagulation, which intentionally ruptures Bruch’s membrane (Shah et al., 2015). A major advantage of the single-hit laser-induced CNV model is its ability to reproducibly generate focal neovascular lesions within a short, well-defined time course, in contrast to spontaneous CNV development in transgenic models (Lambert et al., 2013; Pennesi et al., 2012). This approach also permits the use of widely available and cost-effective animals, typically pigmented C57BL/6J mice aged 6–8 weeks, thereby supporting reproducibility across laboratories. Following laser injury, the model reliably produces focal CNV lesions that can be quantified using histological, angiographic, and molecular endpoints (Lambert et al., 2013; Pennesi et al., 2012; Grossniklaus et al., 2010; Poor et al., 2014). Sample collection should be matched to the endpoint being evaluated. CNV area and vascular morphology are typically assessed in RPE/choroid or RPE/choroid/sclera flat mounts, whereas molecular analyses of angiogenic, inflammatory, complement, extracellular matrix, or fibrotic pathways are commonly performed using the RPE/choroid complex, where the laser-induced lesion forms. In contrast, retinal tissue or retinal cross-sections are more appropriate for evaluating secondary retinal responses, including photoreceptor disruption, Müller glial activation, retinal edema, or neuroinflammatory changes.

Fig. 4. — Single-hit laser photocoagulation murine model of choroidal neovascularization (CNV).

Schematic overview of the experimental workflow and pathological features of the single-hit laser-induced CNV model in mice. (1) Anesthetized C57BL/6J mice are positioned on a retinal imaging platform, such as the Micron IV system, to allow real-time fundus visualization. (2) Using fundus guidance, focal laser photocoagulation is applied at four cardinal positions surrounding the optic nerve head to induce controlled rupture of Bruch’s membrane. Successful laser injury initiates a wound-healing response characterized by disruption of the RPE and underlying choroid. (3) CNV development and lesion activity are assessed using multimodal retinal imaging, including fluorescein angiography to evaluate vascular leakage and OCT to visualize structural changes within the outer retina. Laser-induced injury triggers robust angiogenic and inflammatory signaling, leading to the formation of neovascular complexes extending from the choroid into the subretinal space. Upregulation of pro-angiogenic mediators, including VEGF family members, angiopoietin-2, PDGF-BB, bFGF, TGF-β, HGF, IGF-1, inflammatory cytokines, and matrix-remodeling enzymes such as MMP-2 and MMP-9, supports endothelial proliferation, vascular permeability, and lesion expansion. The illustration was created using BioRender. CNV, choroidal neovascularization; RPE, retinal pigment epithelium; OCT, optical coherence tomography; VEGF, vascular endothelial growth factor; PDGF-BB, platelet-derived growth factor-BB; bFGF, basic fibroblast growth factor; TGF-β, transforming growth factor-beta; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; MMP, matrix metalloproteinase; MMP-2, matrix metalloproteinase-2; MMP-9, matrix metalloproteinase-9.

In this model, laser lesions are commonly generated using a green argon laser (532 nm) with pulse durations of approximately 70 ms and power settings ranging from 180 to 360 mW (Gong et al., 2015). Lesions are typically applied at four cardinal positions around the optic nerve head using a retinal imaging system such as the Micron IV, which provides real-time fundus visualization to ensure accurate targeting and helps minimize inadvertent damage to retinal vessels. Lesions are generally spaced at a distance approximately twice the diameter of the optic nerve to prevent lesion fusion. Successful rupture of Bruch’s membrane is confirmed by the appearance of a transient vaporization bubble immediately following laser application or by early structural changes visualized by OCT within the first 24–48 h after injury (Lambert et al., 2013; Poor et al., 2014; Giani et al., 2011).

Laser wavelength selection can influence the extent of retinal damage and the associated wound-healing response. Infrared or red lasers (650–810 nm) are absorbed predominantly by melanin within the RPE-choroid complex and penetrate more deeply into the inner choroid, whereas green lasers (532 nm) are also strongly absorbed by hemoglo bin and tend to produce more superficial photothermal injury involving the outer retina (Mainster, 1986). As a result, laser-induced CNV may also engage retinal wound-healing pathways, including reactive gliosis. Under physiological conditions, Müller glial cells regulate neurotransmitter homeostasis and provide metabolic support to retinal neurons. Following photoreceptor injury, Müller glia undergo reactive gliosis characterized by increased expression of intermediate filaments, a response that can progress to glial scar formation and recapitulate selected pathological features observed in advanced nAMD (Graca et al., 2018; Bringmann et al., 2006, 2012).

Despite its widespread use, the single-hit laser-induced CNV model has important limitations that should be considered when interpreting experimental outcomes. This model should be regarded primarily as an acute injury-induced neovascular model rather than a fully chronic model of AMD (Lambert et al., 2013; Shah et al., 2015). Laser rupture of Bruch’s membrane initiates an early wound-healing response characterized by inflammation, vascular leakage, and angiogenesis, whereas later time points may show lesion remodeling, partial regression, and, in some settings, limited chronic-like sequelae such as fibrosis or scar formation (Horrigan et al., 2026; Lambert et al., 2013). Thus, the acute and chronic features of this model are sequential rather than simultaneous. Accordingly, it is well suited for investigating CNV induction, early angiogenic responses, vascular leakage, lesion progression, and short-term therapeutic efficacy in neovascular AMD, but it does not capture the slow, multifactorial pathogenesis of dry AMD. In addition, because lesion formation is induced by acute mechanical injury, the model does not fully reproduce the spontaneous disease evolution observed in human AMD. Not all laser-induced lesions accurately reflect clinically relevant CNV and therefore require exclusion from the analysis based on predefined quality-control criteria (Supplementary Table 1). Although the single-hit laser CNV model does not recapitulate the full spectrum of AMD pathology, it remains one of the most robust and reproducible experimental systems for studying CNV initiation, lesion progression, and therapeutic response (Shah et al., 2015). When applied with appropriate rigor and exclusion criteria, this model continues to provide valuable insight into neovascular mechanisms relevant to advanced AMD.

4.2. Double-hit laser-induced subretinal fibrosis in mice

Subretinal fibrosis represents a terminal pathological outcome of nAMD and is clinically observed as a dense fibrovascular plaque or disciform scar that disrupts the precise laminar organization of the retina (Tenbrock et al., 2022; Armendariz et al., 2024). This fibrotic remodeling occurs downstream of CNV and results in irreversible damage to photoreceptors, the RPE, and Bruch’s membrane (Tenbrock et al., 2022; Hwang et al., 2011). Because subretinal fibrosis is not adequately reproduced by the single-hit laser-induced CNV model, a two-stage laser injury paradigm, commonly referred to as the double-hit laser model, has been developed to more reliably induce fibrovascular scarring in vivo (Fig. 5) (Little et al., 2020). In this approach, laser photocoagulation is used to generate controlled, localized injury to the posterior pole of the eye, thereby triggering a wound-healing response that progresses toward fibrosis (Tenbrock et al., 2022). Accordingly, this model is particularly useful for studying late-stage fibrovascular remodeling, profibrotic signaling pathways, and antifibrotic therapeutic strategies, rather than early angiogenic events or primary CNV induction.

Fig. 5. — Double-hit laser photocoagulation murine model of subretinal fibrosis.

Schematic representation of the experimental design and fibrotic mechanisms induced by the double-hit laser photocoagulation model used to study subretinal fibrosis in neovascular AMD. (1) Anesthetized C57BL/6J mice are positioned on a retinal imaging platform, such as the Micron IV system, to enable precise, fundus-guided laser delivery. (2) An initial series of focal laser photocoagulation lesions is applied at four cardinal positions surrounding the optic nerve head to induce controlled injury to Bruch’s membrane and the retinal pigment epithelium (RPE). (3) Ten days after the initial injury, a second laser application is delivered to the same lesion sites, reinforcing tissue disruption and initiating a sustained wound-healing response that favors fibrotic remodeling. (4) Retinal imaging modalities, including fluorescein angiography and OCT, are used to monitor lesion activity, vascular leakage, and progressive structural remodeling. Repeated laser injury promotes separation of the RPE from underlying tissues and the formation of a subretinal space enriched in profibrotic signaling. Injured RPE and endothelial cells secrete mediators such as TGF-β and VEGF, driving epithelial-to-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition (EndMT), respectively. These processes are accompanied by myofibroblast differentiation, extracellular matrix deposition, and activation of innate immune cells, including macrophages and microglia, thereby amplifying inflammatory and profibrotic signaling. Together, these events culminate in the formation of fibrovascular lesions that recapitulate key features of subretinal fibrosis observed in advanced neovascular AMD. The illustration was created using BioRender. AMD, age-related macular degeneration; CNV, choroidal neovascularization; RPE, retinal pigment epithelium; OCT, optical coherence tomography; TGF-β, transforming growth factor-beta; VEGF, vascular endothelial growth factor; EMT, epithelial-to-mesenchymal transition; EndMT, endothelial-to-mesenchymal transition; ECM, extracellular matrix.

In the double-hit model, pigmented C57BL/6J mice are anesthetized and subjected to an initial series of laser photocoagulation lesions applied at four cardinal positions surrounding the optic nerve head (Little et al., 2020). Lesions are generated using defined laser parameters that induce focal injury without causing overt rupture of retinal blood vessels, similar to the single-hit CNV protocol. Approximately ten days after the initial injury, a second laser exposure is applied directly to the original lesion sites using identical settings. Ocular tissues are subsequently harvested at defined intervals, typically 4 to 30 days after the second laser application, to assess fibrotic progression. Mechanistically, repeated laser injury disrupts the integrity of Bruch’s membrane and promotes separation between the RPE and underlying tissues, creating a subretinal microenvironment that favors EMT, EndMT, myofibroblast differentiation, and extracellular matrix deposition (Fig. 5) (Ishikawa et al., 2016; Grossniklaus et al., 2010; Hwang et al., 2011). Injured RPE cells secrete profibrotic mediators, including TGF-β and VEGF, which cooperatively reinforce EMT and EndMT programs (Grossniklaus et al., 2010; Saika et al., 2009). Concurrently, activation of innate immune cells such as macrophages and microglia amplifies inflammatory and profibrotic cytokine production, further driving extracellular matrix deposition and scar formation. Fibrotic lesions are detected clinically as areas of hyperpigmentation on fundoscopic examination, while lesion size, depth, and structural remodeling are quantified using OCT on imaging platforms such as the Micron IV system (Combadiere et al., 2007; Giani et al., 2011; Saika et al., 2009). Sample collection should be specified according to whether the endpoint reflects fibrotic lesion morphology, molecular profibrotic signaling, or secondary retinal injury. Subretinal fibrosis and fibrovascular scar formation are commonly assessed in RPE/choroid or RPE/choroid/sclera flat mounts and in retinal/RPE-choroid cross-sections using markers of extracellular matrix deposition and myofibroblast differentiation. Molecular analyses of EMT, EndMT, inflammatory, angiogenic, and profibrotic pathways are typically most informative in the RPE/choroid complex, where repeated laser injury and fibrovascular lesions develop. In contrast, retinal tissue or retinal sections are more appropriate for evaluating secondary photoreceptor disruption, retinal gliosis, or neurodegenerative changes overlying the fibrotic lesion. Despite its utility, the double-hit laser model shares limitations common to injury-based murine systems. It does not fully recapitulate the complex temporal and spatial progression of fibrosis observed in human AMD, nor does it capture the gradual transition from early retinal dysfunction to end-stage fibrovascular scarring. Instead, it represents a defined, experimentally induced late-stage injury paradigm that isolates fibrotic mechanisms at a specific point in disease progression (Pennesi et al., 2012; Marmorstein et al., 2007). Although this model is more suitable than the single-hit laser CNV model for studying subretinal fibrosis, it remains less informative for investigating the earliest pathogenic events that drive AMD onset. Furthermore, the molecular heterogeneity underlying distinct subtypes of subretinal fibrosis remains incompletely understood. Nevertheless, double-hit laser models remain valuable tools for interrogating fibrotic mechanisms and evaluating therapeutic strategies to limit scar formation and preserve vision in late-stage neovascular disease.

4.3. Spontaneous CNV transgenic model: JR5558

The progressive development of subretinal fibrosis is challenging to recapitulate in murine models, particularly given the invasive nature of injury-based paradigms and the abrupt onset of post-injury wound-healing responses. In human nAMD, fibrosis typically develops downstream of vascular leakage and hemorrhage, progressing toward retinal atrophy and formation of a subretinal fibrotic scar (Friedlander, 2007; Romano et al., 2023). In spontaneous neovascular models, the JR5558 mouse exhibits early-onset, bilateral, multifocal subretinal lesions with progressive inflammatory, angiogenic, and fibrovascular remodeling in the absence of laser-induced injury (Nagai et al., 2014; Chang et al., 2018; Seyed-Razavi et al., 2024; Hasegawa et al., 2014). In contrast to acute injury-based paradigms, JR5558 enables longitudinal investigation of spontaneous lesion evolution, from initial neovascularization to later remodeling, within a relatively defined and experimentally tractable time window (Nagai et al., 2014; Hasegawa et al., 2014). The model is further characterized by its reproducibility, quantifiable lesion burden, and reported partial responsiveness to anti-VEGF therapy, allowing interrogation of both treatment-sensitive and incompletely responsive disease mechanisms (Nagai et al., 2014; Hasegawa et al., 2014; Foxton et al., 2019). Compared with other spontaneous models, such as the VLDLR knockout mouse, which more specifically reflects retinal angiomatous proliferation (RAP)-like pathology, JR5558 provides a complementary platform for studying chronic inflammatory-neovascular interactions and fibrovascular lesion progression relevant to late-stage nAMD (Nagai et al., 2014; Hasegawa et al., 2014). JR5558 recapitulates spontaneous lesion development, immune activation, and progressive remodeling, making it a valuable experimental system for investigating selected aspects of disease progression and therapeutic response in neovascular AMD. Thus, it is discussed here as a representative and experimentally tractable model for investigating chronic neovascular-fibrotic mechanisms and treatment response without the confounding effects of laser injury.

JR5558 mice exhibit early-onset retinal pathology, with subretinal lesions emerging between postnatal days 21 and 30. By three to four weeks of age, these mice develop yellow subretinal lesions beneath the neural retina that increase in number and size, reaching peak severity between four and eight weeks (Seyed-Razavi et al., 2024). OCT reveals corresponding regions of subretinal hyperreflectivity, allowing longitudinal quantification of lesion size, area, and spatial distribution. Neovascular tufts arise from the choriocapillaris and extend toward the midperipheral fundus, with lesion counts reaching approximately 15–20 per eye by postnatal day 30 (Chang et al., 2018). These features distinguish JR5558 from acute-injury-based CNV models by providing a spontaneous, progressive lesion phenotype while retaining sufficient reproducibility for experimental analysis.

In JR5558 mice, the sample source should be specified because spontaneous lesions involve both the subretinal/RPE-choroid interface and secondary changes in the neural retina. Lesion number, lesion area, vascular morphology, and fibrovascular remodeling are commonly assessed using RPE/choroid or RPE/choroid/sclera flat mounts and retinal/RPE-choroid cross-sections. Molecular and histological analyses of angiogenesis, fibrosis, EMT, complement activation, and immune-cell infiltration are most informative when localized to the RPE/choroid complex, subretinal lesion tissue, or retinal sections. In contrast, retinal tissue or retinal sections should be specified when evaluating photoreceptor degeneration, Müller glial activation, retinal edema, or inner- and outer-retinal microglial responses. Histological and immunohistochemical analyses demonstrate that these lesions are characterized by extracellular matrix accumulation and fibrovascular remodeling. Increased collagen deposition is evident by hematoxylin and eosin and Picro-Sirius Red staining, while immunolabeling reveals angiogenic and fibrotic markers, including alpha-smooth muscle actin (α-SMA), collagen IV, and glial fibrillary acidic protein (GFAP), indicating concurrent angiogenesis and gliosis. Connective tissue growth factor (CTGF), a key regulator of proliferation, wound healing, angiogenesis, and fibrosis, is markedly upregulated in the outer retina of JR5558 mice compared with wild-type C57BL/6 controls (Ishikawa et al., 2016; Seyed-Razavi et al., 2024).

Immunofluorescence studies further reveal that neovascular growth in JR5558 mice originates from both the retinal vascular plexus and the choriocapillaris, with vessels penetrating the RPE. Müller glial activation is prominent, with reactive processes visualized by vimentin and cellular retinaldehyde-binding protein (CRALBP), a marker expressed in both Müller cells and the RPE (Chang et al., 2018; Seyed-Razavi et al., 2024). Fibrovascular subretinal deposits display elevated expression of α-SMA, glutamate-aspartate transporter (GLAST), collagen I, collagen IV, fibronectin, CTGF, and markers of gliosis, consistent with active fibrotic remodeling (Seyed-Razavi et al., 2024; Qiang et al., 2021). Structural integrity of the RPE is progressively compromised in JR5558, with RPE cytoskeletal disruption detectable by phalloidin staining at later stages, accompanied by focal photoreceptor disorganization and loss (Seyed-Razavi et al., 2024). Photoreceptor integrity is similarly affected, with immunostaining for cone arrestin and rhodopsin showing focal disorganization and loss of both rod and cone photoreceptors. Immune cell infiltration is a defining feature of this model. Iba1-positive microglia accumulate within the inner plexiform layer and outer plexiform layer, while amoeboid microglia infiltrate the subretinal space, reflecting chronic immune activation. Notably, JR5558 mice exhibit partial responsiveness to anti-VEGF therapy (Foxton et al., 2019). Treatment with aflibercept at four weeks of age reduces lesion growth and outer retinal abnormalities, although the magnitude of response is modest compared with controls (Seyed-Razavi et al., 2024). This partial therapeutic responsiveness may increase the translational relevance of JR5558 for studying treatment-resistant or incompletely responsive neovascular-fibrotic disease states.

Genetically, JR5558 mice harbor autosomal recessive mutations in crumbs homolog 1 retinal degeneration 8 (Crb1rd8) and janus kinase 3, mutation 1 (JAK3M1J), which together contribute to persistent retinal edema, increased vascular permeability, focal gliosis, spontaneous neovascularization of choroidal origin, and progressive photoreceptor degeneration (Chang et al., 2018; Mattapallil et al., 2012). Fundoscopic examination reveals consistent retinal depigmentation, a feature that may exacerbate oxidative stress, given melanin’s role in absorbing light-generated reactive oxygen species (Seyed-Razavi et al., 2024; Sarna, 1992). Mechanistic studies in this model have focused on identifying the cellular origins of myofibroblasts involved in subretinal fibrosis. Candidate progenitor populations include RPE cells, Müller glia, vascular pericytes, and infiltrating macrophages, with epithelial-to-mesenchymal transition (EMT) representing a shared pathway (Saika et al., 2009; Tamiya et al., 2016). RPE cells in JR5558 mice exhibit EMT-associated signaling, including activation of TGFβ, SMAD2, MAPK, and MRTF pathways, accompanied by disruption of tight junction integrity, as indicated by altered zonula occludens-1 (ZO-1) localization (Saika et al., 2009). Because the subretinal space is normally immune-privileged, the presence of chronically activated microglia, complement activation, and infiltrating choroidal macrophages provides a potent stimulus for fibrotic remodeling. Current evidence supports a model in which leukocyte extravasation through immature, permeable neo vessels precedes the macrophage-to-myofibroblast transition, driven in part by complement component 3a (C3a) and TGF-β signaling (Little et al., 2018; Nozaki et al., 2006; Meng et al., 2016). Ongoing studies aim to clarify whether these fibrogenic macrophages arise predominantly from yolk sac-derived microglia or from circulating monocyte-derived macrophages (Sennlaub et al., 2013; Ginhoux et al., 2010).

Despite its strengths, the JR5558 model has important limitations. Although it offers advantages over injury-based models by enabling spontaneous lesion development and chronic inflammatory-fibrotic progression, its phenotype is driven by specific recessive mutations that do not directly mirror the genetic architecture of most human AMD cases (Nagai et al., 2014; Hasegawa et al., 2014). In addition, compared with other spontaneous CNV models, such as the very low density lipoprotein receptor (VLDLR) knockout mouse, JR5558 lesions are less visually apparent at later stages, which may limit direct comparison with more overt neovascular phenotypes (Heckenlively et al., 2003). As with all murine AMD models, the absence of a macula constrains anatomical fidelity, and isolation of pure RPE-derived protein remains technically challenging. Additionally, most studies have been conducted in male mice, leaving the influence of biological sex and sex hormones on lesion development largely unexplored. Nevertheless, the spontaneous emergence of fibrovascular subretinal lesions in JR5558 mice provides a valuable platform for dissecting chronic inflammatory, angiogenic, and fibrotic mechanisms relevant to late-stage AMD. Further characterization of the genetic drivers of this phenotype may facilitate the development of targeted therapeutic strategies to delay or prevent subretinal fibrosis.

4.4. Anti-VEGF resistant CNV model

The term anti-VEGF-resistant model refers specifically to an aged laser-induced CNV paradigm in which CNV lesions generated in aged mice show reduced responsiveness to anti-VEGF monotherapy compared with lesions generated in young adult mice. This model was developed to investigate incomplete therapeutic response in neovascular age-related macular degeneration, in which some patients exhibit persistent exudation, reduced anatomical improvement, or a declining response despite continued anti-VEGF treatment. Anti-VEGF therapy remains the standard treatment for neovascular AMD, as established by landmark clinical trials of ranibizumab, bevacizumab, and related VEGF-targeting agents (Rosenfeld et al., 2006; Comparison of Age-related Macular Degeneration Treatments Trials Research and G., 2012). Unlike conventional laser-induced CNV in young mice, which is largely VEGF-responsive, the aged paradigm introduces therapeutic resistance as a measurable experimental endpoint (Pennesi et al., 2012; Zhu et al., 2020; Zhang et al., 2022).

The construction of this paradigm follows the standard laser photocoagulation approach used to rupture Bruch’s membrane and induce CNV, but the critical modification is the use of aged animals, commonly around 18 months of age. In preclinical studies comparing young, middle-aged, and old mice after laser injury and intravitreal anti-VEGF treatment, CNV in young mice was readily suppressed by anti-VEGF therapy, whereas lesions in old mice showed a poor response, even when the anti-VEGF antibody dose was increased fivefold (Zhu et al., 2020). This age-dependent reduction in drug responsiveness provided the operational basis for defining aged laser-induced CNV as an anti-VEGF-resistant paradigm.

Phenotypically, this model differs from standard young-mouse laser CNV not only by treatment response but also by vascular architecture. Subsequent work further refined the paradigm by distinguishing capillary CNV from arteriolar CNV. Young mice predominantly developed capillary-like lesions that remained VEGF-sensitive, whereas aged mice developed arteriolarized lesions characterized by larger-caliber vessels, branching arterioles, feeder vessels, vascular loops, increased vascular complexity, and greater leakage. These lesions were poorly suppressed by aflibercept alone, while combination therapy with AIBP, apoA-I, and aflibercept inhibited the resistant arteriolar phenotype (Zhang et al., 2022).

The model is assessed using complementary structural and functional readouts. Fluorescein angiography is used to evaluate leakage and hyperpermeability; indocyanine green angiography helps identify arteriolar structures, feeder vessels, and vascular loops; optical coherence tomography provides in vivo assessment of lesion morphology; and isolectin-labeled RPE/choroid flat mounts are used to quantify CNV area and classify vascular morphology (Fu et al., 2024; Zhang et al., 2022). Thus, the defining endpoint is not simply CNV formation but the persistence of CNV despite anti-VEGF treatment, together with arteriolar vascular features associated with reduced therapeutic responsiveness (Fu et al., 2024; Zhang et al., 2022). Because this paradigm is defined by both treatment response and lesion architecture, interpretation of experimental findings depends on the ocular compartment analyzed. RPE/choroid or RPE/choroid/sclera flat mounts are most appropriate for quantifying the area of resistant CNV and arteriolar vascular morphology, whereas the RPE/choroid complex is most relevant for molecular analyses of macrophage activation, lipid accumulation, cholesterol efflux, inflammatory signaling, and angiogenic remodeling. Retinal tissue or retinal sections are more appropriate for evaluating secondary retinal injury, photoreceptor disruption, gliosis, or neuroinflammatory responses associated with persistent CNV.

Biologically, the paradigm is significant because it links anti-VEGF resistance to age-associated changes in the inflammatory and metabolic microenvironment of CNV lesions. Experimental work showed that reduced anti-VEGF efficacy in aged mice correlated with increased lipid accumulation in macrophages, and that macrophage depletion restored anti-VEGF sensitivity (Zhu et al., 2020). These findings suggest that resistance in this model is not merely due to insufficient VEGF blockade, but reflects a shift toward macrophage-associated, cholesterol-linked, and partially VEGF-independent vascular remodeling. This interpretation is consistent with broader evidence that activated macrophages and myeloid cell populations contribute to the biology of neovascular AMD and to experimental CNV formation (Hagbi-Levi et al., 2021; Droho et al., 2023; Andriessen et al., 2021).

Mechanistically, AIBP/apoA-I therapy is proposed to overcome resistance by enhancing cholesterol efflux from activated macrophages and endothelial cells, thereby reducing lipid-raft-dependent inflammatory and angiogenic signaling. When combined with anti-VEGF therapy, this strategy targets both VEGF-dependent capillary angiogenesis and macrophage-associated arteriolar remodeling. The success of AIBP, apoA-I, and aflibercept in suppressing resistant lesions supports the broader concept that anti-VEGF-resistant CNV may require combination approaches that address both angiogenesis and inflammatory vascular remodeling (Fu et al., 2024; Zhu et al., 2020; Zhang et al., 2022).

Despite its utility, the aged anti-VEGF-resistant laser-CNV paradigm has important limitations. It remains an injury-induced model and does not reproduce the slow, multifactorial development of human neovascular AMD. In addition, resistance is modeled functionally as a poor response to anti-VEGF treatment in aged mice, rather than as chronic, repeated exposure to anti-VEGF over months or years. The model also does not capture the full heterogeneity of clinical nonresponse, which may involve lesion subtype, fibrosis, tachyphylaxis, pharmacokinetics, genetic background, and coexisting retinal pathology (Pennesi et al., 2012; Fu et al., 2024; Zhu et al., 2020; Zhang et al., 2022). Therefore, this paradigm is best used to study age-associated, macrophage-linked, arteriolar CNV with reduced anti-VEGF responsiveness, rather than as a complete model of all forms of clinical anti-VEGF resistance.

4.5. VLDLR knockout model

VLDLR is a multifunctional member of the low-density lipoprotein receptor family that binds apolipoprotein E (apoE)-containing lipoproteins, including very low-density lipoproteins (VLDL) and intermediate-density lipoproteins (IDL), as well as additional ligands such as lipoprotein lipase (LPL), lipoprotein(a), proprotein convertase subtilisin/kexin type 9 (PCSK9), urokinase plasminogen activator (uPA), and tissue factor pathway inhibitor (TFPI) (Takahashi et al., 2004; Willnow et al., 1999; Poirier et al., 2008).

Beyond its metabolic role, VLDLR has been extensively studied in the eye for its function in regulating angiogenesis and neuronal organization. VLDLR knockout (VLDLR^−/−) mice are best regarded as a spontaneous model of retinal angiomatous proliferation (RAP)-like or type 3 neovascularization-like pathology, rather than as a generic model of classic choroidal neovascularization (Heckenlively et al., 2003; Hu et al., 2008). This classification is based on the characteristic origin of abnormal vessels from the retinal circulation, with extension from the outer plexiform layer into the subretinal space and subsequent formation of retinal-choroidal anastomoses (Heckenlively et al., 2003; Hu et al., 2008). Thus, although the model falls within the broader spectrum of neovascular AMD-related animal models, it more specifically represents retinally derived neovascularization than conventional type 1 or type 2 CNV arising primarily from the choroid (Heckenlively et al., 2003; Qiang et al., 2021; Hu et al., 2008).

This distinction is important for determining the model’s applicability. The VLDLR^−/− mouse is appropriately classified within the broader category of neovascular AMD models because it recapitulates spontaneous subretinal neovascularization, exudative remodeling, photoreceptor dysfunction, and late fibrovascular changes in the absence of laser injury. However, it should not be generalized as a universal CNV model without qualification, because its vascular origin, lesion architecture, and disease course preferentially reflect RAP/type 3-like biology. Accordingly, this model is particularly useful for investigating retinal-choroidal anastomosis, immune-vascular interactions, cone vulnerability, lipid-associated vascular dysregulation, and angiogenic remodeling in a chronic, non-injury-based setting. Because VLDLR^−/− lesions arise predominantly from the retinal vasculature and extend into the subretinal space, interpretation of experimental findings depends strongly on the ocular compartment analyzed. Retinal tissue and retinal cross-sections are most appropriate for assessing retinal vascular tufts, outer plexiform layer involvement, photoreceptor degeneration, cone dysfunction, Müller glial responses, and retinal microglia/macrophage accumulation. RPE/choroid or RPE/choroid/sclera preparations are more relevant when evaluating retinal-choroidal anastomoses, subretinal fibrovascular remodeling, or secondary changes at the RPE-choroid interface. Therefore, molecular or histological findings from VLDLR^−/− mice should be interpreted based on whether the sample represents the retina, RPE/choroid, the combined posterior eyecup, or lesion-localized tissue.

By postnatal day 14, VLDLR^−/− mice exhibit abnormal vascular growth extending from the outer plexiform layer into the subretinal space, accompanied by progressive photoreceptor degeneration and thinning of the outer nuclear layer (Heckenlively et al., 2003; Hu et al., 2008; Jiang et al., 2009). With disease progression, extensive vascular infiltration of the subretinal space leads to fibrovascular remodeling and subretinal fibrosis. These pathological changes are thought to arise from dysregulated Wnt-dependent endothelial migration and proliferation in the absence of VLDLR-mediated inhibitory signaling (Hu et al., 2008).

Electroretinographic analyses reveal that photoreceptor dysfunction in VLDLR^−/− mice is particularly pronounced in cone photoreceptors, a notable feature given the cone-rich composition of the human macula, which is responsible for high-acuity and color vision (Hu et al., 2008; Xia et al., 2011). Studies by Friedlander and colleagues further demonstrated robust colocalization of activated microglia and macrophages with subretinal neovascular tufts in VLDLR^−/− mice, as visualized by confocal microscopy and indocyanine green angiography (Usui-Ouchi et al., 2020).

An additional hallmark of the VLDLR knockout model is the formation of retinal-choroidal anastomoses, resembling the arteriovenous shunts characteristic of arteriolar CNV (Heckenlively et al., 2003). These vascular abnormalities further underscore the relevance of the VLDLR^−/− mouse as a model of VEGF-independent pathological angiogenesis (Heckenlively et al., 2003; Hu et al., 2008). These vascular abnormalities, together with later fibrovascular remodeling and fibrosis by 3 to 4 months of age, make the VLDLR^−/− mouse particularly informative for studying spontaneous lesion progression and late-stage neovascular remodeling (Hu et al., 2008; Xia et al., 2011).

Despite its utility, the VLDLR knockout model has important limitations. Its phenotype is driven by global loss of VLDLR function, such that systemic lipid dysregulation and developmental vascular effects may influence ocular pathology in ways that do not fully reflect the multifactorial etiology of human AMD (Heckenlively et al., 2003; Hu et al., 2008; Xia et al., 2011). More importantly, because the neovascular lesions arise predominantly from the retina rather than the choroid, generalizing this model as a standard CNV model may lead to overinterpretation of findings across all neovascular AMD subtypes. Conclusions drawn from VLDLR^−/− mice are therefore most appropriately applied to RAP/type 3-like neovascularization and related retinal-choroidal vascular remodeling, rather than assumed to represent the full biology of classic CNV. As with all murine AMD models, the absence of a macula limits anatomical fidelity. Nevertheless, the VLDLR^−/− mouse remains a valuable model for studying spontaneous neovascularization, cone vulnerability, immune-vascular interactions, lipid-associated angiogenesis, and late fibrovascular remodeling within the specific context of RAP/type 3-like disease (Nozaki et al., 2006; Sene et al., 2013; Pennesi et al., 2012; Xia et al., 2011).

4.6. CYP27A1 knockout model

Neovascular AMD encompasses several subtypes of CNV, including type 1 (sub-RPE proliferation), type 2 (classic subretinal CNV), and type 3 CNV, also referred to as retinal angiomatous proliferation (RAP), which is characterized by aberrant vascular remodeling and retinal-choroidal vascular connections (Freund et al., 2008). Cytochrome P450 27A1 (CYP27A1) is a mitochondrial sterol 27-hydroxylase involved in cholesterol metabolism through side-chain oxidation of cholesterol and sterol intermediates, contributing to cholesterol elimination via oxysterol formation. In the retina, CYP27A1 participates in cholesterol turnover alongside CYP46A1, and disruption of these pathways alters retinal cholesterol homeostasis (Mast et al., 2011). Disruption of cholesterol homeostasis has been implicated in pathological angiogenesis associated with these phenotypes (Curcio et al., 2009; Freund et al., 2008). The CYP27A1 knockout (CYP27A1^−/−) mouse recapitulates key aspects of this phenotype, providing a model in which cholesterol dysregulation drives spontaneous neovascularization and retinal-choroidal vascular remodeling (Omarova et al., 2012).

Because CYP27A1^−/− pathology involves both retinal cholesterol dyshomeostasis and RPE/choroid-associated vascular remodeling, interpretation of experimental findings depends on the ocular compartment analyzed. Retinal tissue and retinal sections are most appropriate for assessing OPL disruption, photoreceptor stress, Müller glial activation, hypoxia, and retinal vascular remodeling. RPE/choroid or RPE/choroid/sclera preparations are more relevant for evaluating RPE disruption, lipid deposition, Bruch’s membrane-associated fibrovascular material, choroidal neovascular changes, and retinal-choroidal anastomoses. Molecular analyses of sterol metabolism, inflammatory signaling, and angiogenic remodeling should therefore specify whether the sample represents retina, RPE/choroid, combined posterior eyecup, or lesion-localized tissue.

CYP27A1^−/− mice exhibit early and progressive retinal abnormalities that can be characterized using OCT, histology, and fluorescein angiography. By 1.5 months of age, OCT imaging reveals hyperreflective, whorl-like lesions within the outer plexiform layer (OPL). Histological analyses of older animals demonstrate torsion and disruption of the OPL near the inner nuclear layer interface, accompanied by eosinophilic spherical structures consistent with dilated blood vessels. Fibrovascular material accumulates between Bruch’s membrane and the RPE, disrupting RPE integrity and projecting into the inner and outer photoreceptor segments. These structural changes facilitate aberrant vascular growth from the inner retina through the outer nuclear layer toward the choriocapillaris, consistent with the formation of retinal-choroidal anastomoses, as confirmed by isolectin B4 staining (Federico et al., 2001).

Glial activation is a prominent feature of the CYP27A1^−/− retina. Müller glial cells, identified by glutamine synthase and GFAP expression, exhibit widespread activation, a response typically associated with hypoxia, photoreceptor stress, or retinal injury (Omarova et al., 2012). Fluorescein angiography demonstrates early, irregular hyperfluorescence within the inner nuclear layer and RPE shortly after dye injection, indicating increased vascular permeability and immature vessel formation. Consistent with these findings, anti-pimonidazole staining reveals elevated hypoxic stress throughout the inner segments and RPE of knockout animals, indicating reduced oxygen tension despite preserved baseline retinal function (Omarova et al., 2012).

Lipid dysregulation is a defining feature of this model. Staining with Oil Red O and filipin reveals focal accumulation of neutral lipids and unesterified cholesterol at sites of RPE disruption, often colocalizing with isolectin B4-positive neovascular structures (Omarova et al., 2012). These observations suggest a close spatial relationship between lipid deposition and pathological vascular remodeling. CYP27A1 deficiency impairs mitochondrial side-chain oxidation of cholesterol, thereby reducing the conversion to oxysterol metabolites such as 27-hydroxycholesterol and cholestenoic acids, which normally facilitate cholesterol elimination from retinal tissues (Mast et al., 2011). The latter is a toxic oxysterol generated through nonenzymatic oxidation and has been implicated as a pathogenic mediator in AMD (Rodriguez et al., 2010). Compensatory activation of cholesterol biosynthetic pathways is metabolically demanding and may contribute to local oxygen consumption, potentially exacerbating retinal hypoxic stress in the setting of impaired sterol elimination (Bloch, 1965; Brown et al., 1997).

4.7. Comparative and functional analysis of murine nAMD models

Although no single murine model faithfully recapitulates the full spectrum of human neovascular AMD, each experimental system captures distinct biological processes relevant to specific stages of disease initiation, progression, and therapeutic response. Accordingly, these models should be viewed as complementary rather than interchangeable. A systematic comparison of their molecular drivers, lesion morphology, temporal dynamics, and translational applicability is essential for appropriate experimental design and interpretation (Tables 1–3).

Table 1.

Shared and model-specific molecular signatures in murine nAMD models.

Model	Core upregulated pathways	Angiogenic/Neovascular Phenotype	Fibrotic programs	Metabolic/lipid features	Key distinction

Single-hit laser	VEGF, Ang2, IL-1β, TNFα	Acute, leaky sprouting CNV	Moderate TGF-β	Minimal	Acute injury-driven angiogenesis
Double-hit laser	VEGF + TGF-β	Persistent, expanding CNV with vascular-mesenchymal transition	Strong EMT/EndMT, α-SMA, CTGF	Minimal	Robust fibrovascular scar formation
JR5558	VEGF, CCL2, IL-6	Progressive spontaneous CNV	Progressive fibrosis	Secondary	Chronic inflammatory-fibrotic evolution
Anti-VEGF Resistant CNV	PlGF, FGF2, residual VEGF	VEGF-independent, therapy-refractory CNV	Matrix remodeling	Secondary	Persistent neovascularization despite VEGF blockade
VLDLR^-/-	Wnt/β-catenin	Retinal-choroidal anastomosis/type-3-like NV	Moderate (late)	Lipid dysregulation	Lipid-angiogenesis crosstalk
CYP27A1^-/-	HIF-1α, VEGF	Hypoxia-driven, lipid-associated CNV	Progressive fibrosis	Cholesterol accumulation	Lipid-driven hypoxic angiogenesis

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CNV, choroidal neovascularization; VEGF, vascular endothelial growth factor; Ang2, angiopoietin-2; IL-1β, interleukin-1 beta; TNFα, tumor necrosis factor alpha; TGF-β, transforming growth factor-beta; EMT, epithelial-to-mesenchymal transition; EndMT, endothelial-to-mesenchymal transition; α-SMA, alpha-smooth muscle actin; CTGF, connective tissue growth factor; CCL2, C-C motif chemokine ligand 2 (MCP-1); IL-6, interleukin-6; PlGF, placental growth factor; FGF2, fibroblast growth factor-2; VLDLR, very-low-density lipoprotein receptor; Wnt/β-catenin, Wingless/integrated-beta-catenin signaling pathway; CYP27A1, cytochrome P450 family 27 sub-family A member 1; HIF-1α, hypoxia-inducible factor-1 alpha; NV, neovascularization.

Table 3.

Phenotypic features and experimental context of commonly used murine models of neovascular AMD.

Model	AMD phenotype/subtype represented	Defining phenotype/ primary readouts	Age, sex, and metabolic context	Primary applications (best suited for)	Key limitations	References

Single-hit laser	Acute injury-induced CNV that models neovascular lesion formation rather than early/intermediate dry AMD	• Rapid focal CNV after Bruch’s membrane rupture • •fluorescein leakage • •lesion size by OCT and isolectin B4 flat mounts • •robust early inflammatory and angiogenic signaling	•Lesion severity increases with age •aged mice show larger, more complex lesions and reduced anti-VEGF responsiveness •sex-dependent immune and VEGF-signaling differences may influence variability	•Early angiogenic signaling •vascular permeability •inflammatory triggers, proof-of-concept testing of anti-angiogenic therapies	•Acute mechanical injury •lacks slow multifactorial AMD progression •minimal fibrosis •requires strict lesion exclusion criteria	(Espinosa-Heidmann et al., 2002; Lambert et al., 2013; Gong et al., 2015; Pennesi et al., 2012)
Double-hit laser	Late-stage neovascular AMD with fibrovascular scarring/subretinal fibrosis	•Repeated laser injury yields hyperpigmented fibrotic plaques •ECM deposition and RPE disorganization •OCT-detectable remodeling •increased collagen, α-SMA and CTGF	•Commonly performed in older mice to enhance fibrosis •aging alters macrophage phenotype and wound repair •sex-specific fibrotic outcomes poorly defined	•Subretinal fibrosis •EMT/EndMT mechanism •ECM remodeling, antifibrotic therapeutic strategies	•Experimentally accelerated injury •not informative for disease initiation •fibrosis extent sensitive to timing and age	(Yi et al., 2023; Little et al., 2020)
JR5558	Spontaneous neovascular AMD-like fibrovascular lesion progression	•Early-onset bilateral multifocal subretinal lesions •yellow fundus spots •OCT hyperreflective lesions •ECM deposition, gliosis, RPE disruption, photoreceptor loss and macrophage/microglial infiltration	•Early onset necessitates careful age matching to disease stage •sex effects on immune tone and vascular remodeling remain underexplored	•Chronic lesion evolution, immune-vascular crosstalk, fibrovascular remodeling •partial anti-VEGF responsiveness	•Driven by specific recessive mutations not typical of human AMD •lesion visibility decreases at later stages; no macula	(Nagai et al., 2014; Seyed-Razavi et al., 2024; Foxton et al., 2019)
Anti-VEGF Resistant CNV	Persistent or incompletely responsive neovascular AMD despite VEGF blockade	•Persistent leakage, incomplete regression, arteriolar-like CNV, continued neovascular activity after treatment •typically assessed in treated laser-CNV systems	•Aging promotes larger lesions and reduced treatment responsiveness •immune and metabolic status likely modifies resistance •sex effects insufficiently defined	•Mechanisms of treatment nonresponse •VEGF-independent angiogenesis •combination or second-line therapeutic strategies	•Functional resistance model rather than disease-initiation model •inherits limitations of laser injury	(Fu et al., 2024; Zhang et al., 2022)
VLDLR ^-/-	RAP/type-3-like neovascularization with retinal-origin vessel invasion	•Retinal-derived neovessels extending into subretinal space •retinal-choroidal anastomose •cone dysfunction •microglia-associated neovascular tufts •late fibrovascular remodeling	•Lipid handling, immune activation, and age influence severity •sex-dependent metabolic effects plausible but poorly characterized	•RAP/type-3 biology, immune-vascular interactions •VEGF-independent angiogenesis •cone vulnerability	•Predominantly retinal rather than classic choroidal CNV •global gene deletion introduces systemic metabolic confounds	(Heckenlively et al., 2003; Hu et al., 2008)
CYP27A1 ^-/-	RAP-like neovascular remodeling associated with cholesterol dysregulation	•Early bilateral OCT-visible lesions •pathological retinal/ choroidal neovascularization •lipid and sterol deposition •gliosis, leakage, hypoxia-associated stress	•Sterol metabolism is age- and hormonally regulated •sex- and age- dependent lipid handling may modify inflammation and lesion progression	•Lipid-driven angiogenesis •cholesterol/oxysterol biology •hypoxia-angiogenesis coupling	•Systemic cholesterol metabolism defects •limited generalizability to classic CNV •metabolic confounds require careful interpretation	(Curcio et al., 2009; Saadane et al., 2014; Omarova et al., 2012)

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AMD, age-related macular degeneration; CNV, choroidal neovascularization; RPE, retinal pigment epithelium; OCT, optical coherence tomography; VEGF, vascular endothelial growth factor; Ang2, angiopoietin-2; IL-1β, interleukin-1 beta; TNFα, tumor necrosis factor alpha; TGF-β, transforming growth factor-beta; ECM, extra-cellular matrix; α-SMA, alpha-smooth muscle actin; CTGF, connective tissue growth factor; EndMT, endothelial-to-mesenchymal transition; EMT, epithelial-to-mesenchymal transition; RAP, retinal angiomatous proliferation; VLDLR, very-low-density lipoprotein receptor; CYP27A1, cytochrome P450 family 27 subfamily A member 1.

4.7.1. Angiogenic versus fibrotic versus metabolic drivers

Laser-induced CNV models are dominated by acute VEGF-driven angiogenesis and inflammation, whereas spontaneous genetic models incorporate chronic inflammatory, metabolic, and remodeling cues that develop without mechanical injury. The single-hit laser CNV model is therefore optimal for interrogating early angiogenic signaling, vascular permeability, and short-term anti-angiogenic efficacy, but it lacks the chronic inflammatory and fibrotic context of human AMD. In contrast, the double-hit laser model amplifies TGF-β–driven EMT/EndMT programs and extracellular matrix deposition, creating a fibrovascular phenotype more relevant to end-stage scarring but less informative about disease initiation.

Spontaneous models such as JR5558, VLDLR^−/− and CYP27A1^−/− introduce persistent inflammatory and metabolic stressors. JR5558 uniquely combines chronic inflammation, gliosis, neovascularization, and progressive fibrosis, making it well-suited for studying lesion evolution and partial anti-VEGF responsiveness. VLDLR and CYP27A1 - models preferentially reflect RAP/type 3-like neovascularization, emphasizing retinal-derived angiogenesis, lipid dysregulation, and VEGF-independent vascular remodeling rather than classic choroidal CNV.

4.7.2. Distinct CNV morphologies across models

CNV architecture differs fundamentally across experimental systems (Table 2). Laser-induced lesions are focal, vertically oriented, and initiated by direct rupture of Bruch’s membrane, whereas spontaneous models generate multifocal, horizontally expanding vascular networks with mixed retinal and choroidal origins. RAP-like models are distinguished by intraretinal vascular proliferation and retinal–choroidal anastomoses, accompanied by arteriolar remodeling that is comparatively resistant to VEGF blockade. These structural differences are not merely anatomical but reflect distinct angiogenic mechanisms and therapeutic vulnerabilities.

Table 2.

CNV morphology differences across murine models.

Model	Vessel origin	Common Tissue(s) Collected	Disease course

Single-hit laser	Choroid	RPE + choroid	Acute
Double-hit laser	Choroid	RPE + choroid	Subacute-chronic
JR5558	Choroid + retina	RPE + choroid (±retina)	Chronic
Anti-VEGF Resistant CNV	Choroid (arteriolar)	RPE + choroid	Chronic
VLDLR^-/-	Retina → subretinal	Retina + RPE + choroid	Chronic
CYP27A1^-/-	Retina → choroid	RPE + choroid	Chronic

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CNV, choroidal neovascularization; RPE, retinal pigment epithelium; VEGF, vascular endothelial growth factor; VLDLR, very-low-density lipoprotein receptor; CYP27A1, cytochrome P450 family 27 subfamily A member 1.

4.7.3. Model-specific applications and limitations

From a translational perspective, model selection should be explicitly guided by the biological question under investigation (Table 3). Laser-based paradigms are most informative for mechanistic dissection of angiogenic triggers and proof-of-concept drug testing, whereas genetic models better approximate chronic disease trajectories, immune–vascular crosstalk, lipid-driven pathology, and incomplete therapeutic responsiveness. Anti-VEGF-resistant paradigms, typically layered onto laser-CNV systems, function as treatment-response models rather than disease-initiation models and are particularly useful for identifying VEGF-independent pathways that sustain persistent neovascularization.

Collectively, systematic comparison across models clarifies that conclusions drawn from one system cannot be indiscriminately generalized. Instead, rational selection and, where appropriate, combined use of complementary models are required to capture the heterogeneous biology underlying neovascular AMD.

5. Age and sex-associated considerations for murine models

Despite substantial progress in identifying genetic and environmental risk factors for neovascular AMD, age- and sex-associated variables remain comparatively understudied in preclinical experimental design, even though evidence indicates that both factors fundamentally shape AMD-relevant biology. Population-based studies and meta-analyses consistently demonstrate steep age-dependent increases in AMD prevalence and report sex-associated differences that vary by disease stage, with several analyses indicating higher prevalence among women (Rudnicka et al., 2012; Collaborators, 2025; Jiang et al., 2023). These observations underscore the importance of incorporating biological sex as a variable in AMD research and highlight the need to better understand how sex hormones, retinal metabolism, immune responses, and therapeutic outcomes differ between males and females.

Beyond epidemiological association, age and biological sex influence multiple pathways implicated in AMD pathogenesis. Sex hormones regulate angiogenesis, oxidative stress responses, inflammatory signaling, and lipid handling (Kaarniranta et al., 2015; Dama et al., 2021; Rowe et al., 2025; Vegeto et al., 2008; Nuzzi et al., 2022; Arevalo et al., 2010). Estrogens, particularly in premenopausal individuals, exert anti-inflammatory, antioxidant, and vasoprotective effects (Dama et al., 2021; Vegeto et al., 2008; Nuzzi et al., 2022; Arevalo et al., 2010). Estrogen receptors (ERα and ERβ) are expressed in retinal pigment epithelial cells and contribute to epithelial stability and cellular homeostasis (Giddabasappa et al., 2010; Elliot et al., 2010; Munaut et al., 2001). Functional studies further demonstrate that estrogen signaling modulates inflammatory pathways in the RPE, including NF-κB-associated signaling cascades, and that loss of ERβ increases susceptibility to RPE injury under environmental stress, supporting a protective role for estrogen signaling in retinal biology (Marin-Castano et al., 2003).

With advancing age and the decline in estrogen levels following menopause, susceptibility to oxidative stress increases, potentially through altered regulation of inflammatory pathways. These hormonal changes may contribute to sex-specific differences in retinal vulnerability and disease progression. Sex-dependent differences also extend beyond hormonal signaling to broader immune regulation. Females generally exhibit more robust innate and adaptive immune responses, which may enhance clearance of cellular debris but can also predispose to sustained inflammatory activation under chronic stress conditions (Klein et al., 2016; Mitchell et al., 2022). Clinical studies have reported age- and sex-associated differences in circulating and ocular complement components among patients with intermediate AMD, indicating biologically distinct inflammatory states within the same clinical stage (Marin et al., 2022). Estrogen signaling also intersects with lipid metabolism and macrophage function. Activation of estrogen receptors enhances cholesterol efflux pathways in macrophages, including ABCA1-dependent lipid transport, thereby influencing foam cell formation and inflammatory tone (Corcoran et al., 2011; Bao et al., 2023). Given that impaired cholesterol handling and lipid accumulation are central features of AMD-relevant immune activation, hormonal regulation of macrophage lipid biology may contribute to sex-specific differences in inflammatory amplification within the subretinal space (Sene et al., 2013; Ban et al., 2018). Declining estrogen levels with age may therefore shift macrophage phenotype toward a more pro-inflammatory or lipid-retentive state, altering the balance between tissue repair and pathologic angiogenesis.

Aging independently reshapes the immune-metabolic environment in which neovascular lesions develop. With advancing age, macrophages exhibit altered polarization dynamics, impaired cholesterol efflux capacity, increased production of proangiogenic mediators, and reduced resolution of inflammation (Sene et al., 2013). Consistent with these changes, aged mice in laser-induced CNV models develop larger, more complex lesions with increased vascular permeability and reduced responsiveness to anti-VEGF therapy compared with young adult mice (Espinosa-Heidmann et al., 2002; Gong et al., 2015; Fu et al., 2024; Zhang et al., 2022). This age-associated shift is further accompanied by changes in lesion morphology, with older animals displaying arteriolar-dominant CNV phenotypes characterized by expansive, confluent neovascularization and widespread fluorescein extravasation, whereas younger mice more commonly exhibit capillary-dominant lesions with discrete borders, reduced leakage, and greater sensitivity to VEGF inhibition (Fu et al., 2024; Zhang et al., 2022). These findings indicate that aging not only increases lesion burden but also fundamentally alters CNV architecture and therapeutic responsiveness, mirroring clinical observations linking arteriolarized or high-flow CNV phenotypes to incomplete responses to anti-VEGF therapy in nAMD (Fu et al., 2024; Spaide, 2015). Importantly, aging also modifies post-angiogenic wound-healing responses and fibrotic remodeling. Fibrotic remodeling, a late consequence of nAMD, is influenced by age-related alterations in extracellular matrix turnover, enhanced TGF-β signaling, and impaired regenerative capacity (Yi et al., 2023; Zhao et al., 2022). In second-hit laser-induced CNV paradigms, aged mice exhibit enhanced subretinal fibrosis characterized by increased collagen deposition, greater recruitment of circulating fibrocytes, and acquisition of a profibrotic macrophage phenotype associated with heightened TGF-β signaling (Yi et al., 2023).

Because estrogen signaling modulates inflammatory activation and matrix deposition pathways, sex-based differences in hormonal regulation may further influence fibrotic severity and scar architecture, although these effects remain incompletely characterized in current experimental systems. Despite the central role of age and sex in shaping AMD-relevant biology, many commonly used murine models rely on young adult animals and frequently employ only a single sex, often without justification or stratified analysis (Espinosa-Heidmann et al., 2002; Lambert et al., 2013; Poor et al., 2014). Single-hit laser-induced CNV studies are typically performed in 6–8-week-old mice to prioritize experimental throughput and reproducibility, despite evidence that both age and sex influence lesion size, variability, and treatment response (Espinosa-Heidmann et al., 2002; Lambert et al., 2013; Gong et al., 2015). While double-hit laser paradigms and aged immune-dysregulation models partially address these limitations by incorporating older animals and enabling the study of subretinal fibrosis (Little et al., 2020), sex-specific effects on fibrotic remodeling, immune activation, and therapeutic resistance remain largely unexplored. Similarly, spontaneous and transgenic models, including JR5558, VLDLR-deficient, and CYP27A1-deficient mice, recapitulate distinct angiogenic, inflammatory, or fibrotic features relevant to AMD (Nagai et al., 2014; Heckenlively et al., 2003; Saadane et al., 2014; Huang et al., 2017).

However, most studies employing these models do not systematically evaluate sex as a biological variable or incorporate age-appropriate cohorts. Given that lipid metabolism, macrophage function, complement activity, and chemokine signaling exhibit well-documented age- and sex-dependent differences, the absence of stratified analyses may obscure critical mechanisms underlying disease heterogeneity and treatment resistance. Moreover, early-onset phenotypes observed in certain genetic models may reflect developmental angiogenic programs rather than aging-associated degeneration, underscoring the importance of interpreting these systems within appropriate age contexts.

Overall, these observations demonstrate that age and biological sex are not merely confounding variables but integral biological determinants of AMD pathophysiology, shaping immune tone, lesion architecture, fibrotic remodeling, and therapeutic responsiveness. Failure to account for these factors in experimental design may obscure meaningful differences in disease mechanisms and limit translational relevance. Incorporating age-appropriate cohorts, ensuring balanced inclusion of both sexes, and conducting predefined age- and sex-stratified analyses in future preclinical studies will be essential for advancing mechanistic understanding and enabling more personalized approaches to AMD management.

6. Future consideration

Given that AMD is fundamentally an age-associated disease, future preclinical studies should systematically incorporate aged animal cohorts rather than relying predominantly on young adult mice. Aging alters immune tone, metabolic capacity, mitochondrial function, and wound-healing responses, all of which influence lesion architecture, therapeutic responsiveness, and disease progression. Models that combine aging with metabolic stress, immune dysregulation, or repeated angiogenic insults may better capture transitions from early degeneration to neovascularization, fibrosis, and treatment resistance. Longitudinal experimental designs that track disease evolution over time, rather than relying on short-term endpoints, will be particularly valuable.

Biological sex should be treated as a core experimental variable rather than a secondary consideration. Future studies should be powered to detect sex-specific differences in immune activation, lipid handling, angiogenic signaling, fibrotic remodeling, and response to therapy. Mechanistic dissection of how sex hormones and sex-linked genetic and metabolic factors shape retinal pathology will be critical for understanding disease heterogeneity and for informing more personalized therapeutic strategies. Reporting sex-disaggregated data should become standard practice in AMD research.

No single murine model recapitulates the full spectrum of human AMD. Future efforts should focus on combining complementary models or developing hybrid paradigms that integrate multiple disease drivers, such as lipid dysregulation, chronic inflammation, immunosenescence, and fibrosis. For example, pairing genetic models of metabolic or immune dysfunction with age, dietary stress, or repeated angiogenic injury may improve modeling of late-stage disease phenotypes. In parallel, refinement of non-murine systems, including human retinal organoids, induced pluripotent stem cell-derived RPE, and ex vivo choroid-RPE platforms, will be essential for bridging species-specific anatomical and metabolic differences.

Future therapeutic strategies for AMD should shift toward early intervention aimed at modifying upstream disease drivers before irreversible retinal damage occurs. Rather than focusing solely on late stage neovascular or atrophic outcomes, targeting early RPE stress, oxidative and metabolic dysfunction, chronic innate immune activation, and dysregulated lipid and complement pathways may reduce progression to advanced disease. This approach is best paired with the development of minimally invasive or noninvasive therapies suitable for long-term use in patients with preserved vision.

Also, therapeutic development must move beyond exclusive reliance on VEGF inhibition. Future strategies should explore pathways implicated in anti-VEGF resistance, including macrophage-driven inflammation, lipid-mediated signaling, complement activation, endothelial metabolic reprogramming, and profibrotic remodeling. Targeting immune-metabolic interfaces, such as cholesterol handling, inflammasome activation, senescence-associated secretory phenotypes, and chemokine signaling, may offer opportunities to modulate disease progression rather than simply suppress angiogenesis. Combination therapies that integrate anti-angiogenic, anti-inflammatory, and antifibrotic mechanisms warrant systematic investigation.

Advancing the understanding and treatment of age-related macular degeneration will require a shift from reductionist approaches toward integrative experimental and translational frameworks that more accurately reflect disease complexity. Although substantial progress has been made in defining individual molecular pathways involved in AMD, future research must prioritize how these pathways interact within the aging retinal microenvironment and across distinct patient subgroups.

7. Conclusion

AMD is a progressive and heterogeneous retinal disease whose prevalence and clinical impact continue to rise with global aging. As outlined in this review, AMD encompasses a spectrum of pathological states, ranging from early drusen accumulation and RPE dysfunction to advanced geographic atrophy and neovascular disease complicated by fibrosis and irreversible vision loss. Although decades of research have substantially advanced understanding of AMD pathophysiology, effective therapeutic interventions remain limited, particularly for dry AMD and for late-stage complications of neovascular AMD, such as treatment resistance and subretinal scarring. These persistent challenges reflect the multifactorial nature of AMD, in which metabolic dysregulation, oxidative stress, chronic inflammation, immune dysfunction, and aberrant angiogenesis converge within an aging retinal environment. These observations underscore the need to move beyond single-pathway frameworks toward integrative models that more faithfully capture disease complexity and progression.

Experimental murine models have been indispensable for advancing mechanistic insight and testing therapeutic strategies, yet no single model fully recapitulates the temporal, cellular, and molecular complexity of human AMD. Acute injury paradigms, such as laser-induced choroidal neovascularization, robustly model angiogenic responses but fail to capture early degenerative events or chronic immune dysregulation. Spontaneous and transgenic systems provide valuable insight into lipid-driven angiogenesis, immune-vascular interactions, and fibrotic remodeling, but often reflect developmental or early-onset phenotypes that differ from those of age-associated disease. Critically, across these models, biological age and sex are often treated as experimental conveniences rather than determinants of disease biology.

The underrepresentation of age-appropriate cohorts and the lack of sex-specific analyses pose a major barrier to translational progress. Accumulating evidence shows that aging alters immune cell function, macrophage and microglial behavior, metabolic stress responses, and angiogenic signaling, while biological sex influences inflammatory tone, vascular remodeling, and therapeutic responsiveness. Failure to incorporate these variables into experimental design may obscure clinically relevant mechanisms and contribute to inconsistent therapeutic outcomes, including anti-VEGF resistance and fibrotic progression.

Future advances in AMD research will depend on developing and applying preclinical models that more faithfully reflect the biological context of human disease. This includes deliberately incorporating aged animals, ensuring balanced representation of both sexes, and integrating immune-metabolic and senescence-related pathways into experimental frameworks. Such approaches will be essential for identifying mechanisms underlying disease heterogeneity and for developing more effective, personalized therapeutic strategies for both dry and neovascular AMD.

Supplementary Material

Supplementary Table

NIHMS2210910-supplement-Supplementary_Table.pdf^{(58.2KB, pdf)}

Acknowledgments

During the preparation of this work, the authors used Co-Pilot (M365) and Grammarly (1.152.1.0) to improve the readability and language of the manuscript. After using this tool, the authors reviewed and edited the text as needed and take full responsibility for the content of the published article. The use of AI generated none of the citations, and the authors take full responsibility for the scientific content in the article.

Funding

The author’s laboratory work is funded by EY034568 (MCT), EY033264 (RNJ & PMM), and EY035336 (RNJ). Student trainees were supported by GM144927, HL007737, and AI007281. Mohammad A. Nisar acknowledges support from the Carolyn L. Luckein Student Research Fellowship program. Chanel Harris acknowledges support from the American Physiological Society (APS) Porter Physiology Development Fellowship.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.exer.2026.111094.

Footnotes

Declaration of competing interest

The authors declare no conflicts of interest.

CRediT authorship contribution statement

Chanel Harris: Writing – original draft, Writing – review & editing. Mohammad A. Nisar: Writing – original draft, Writing – review & editing. Ronny Amamoo: Writing – review & editing. Rahul Patel: Writing – review & editing. Pamela M. Martin: Writing – review & editing. Ravirajsinh N. Jadeja: Writing – review & editing. Menaka C. Thounaojam: Writing – original draft, Writing – review & editing.

Informed consent statement

Not applicable.

Commercial relationships disclosure

None.

Institutional review board statement

Not applicable.

Data availability

No data was used for the research described in the article.

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Supplementary Materials

Supplementary Table

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Data Availability Statement

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PERMALINK

Murine models of neovascular AMD revisited: Mechanistic pathways, immune-metabolic crosstalk, and the impact of aging and sex

Chanel Harris

Mohammad A Nisar

Ronny Amamoo

Rahul Patel

Pamela M Martin

Ravirajsinh N Jadeja

Menaka C Thounaojam

Abstract

1. Introduction

Fig. 1. Major risk factors contributing to age-related macular degeneration (AMD).

2. Types of age-related macular degeneration: wet and dry

2.1. Dry (atrophic) age-related macular degeneration

Fig. 2.

2.2. Wet (neovascular) age-related macular degeneration

3. Cellular and molecular mechanisms in AMD pathogenesis

3.1. Metabolic dysregulation

Fig. 3.

3.2. Oxidative stress

3.3. Inflammation

3.4. Immunosenescence

3.5. Hypoxia signaling

3.6. VEGF signaling

3.7. Extracellular matrix remodeling

4. Experimental murine models for studying nAMD

4.1. Single-hit laser-induced CNV in mice

Fig. 4.

4.2. Double-hit laser-induced subretinal fibrosis in mice

Fig. 5.

4.3. Spontaneous CNV transgenic model: JR5558

4.4. Anti-VEGF resistant CNV model

4.5. VLDLR knockout model

4.6. CYP27A1 knockout model

4.7. Comparative and functional analysis of murine nAMD models

Table 1.

Table 3.

4.7.1. Angiogenic versus fibrotic versus metabolic drivers

4.7.2. Distinct CNV morphologies across models

Table 2.

4.7.3. Model-specific applications and limitations

5. Age and sex-associated considerations for murine models

6. Future consideration

7. Conclusion

Supplementary Material

Acknowledgments

Funding

Appendix A. Supplementary data

Footnotes

Data availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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