Age-related macular degeneration (AMD) is a leading cause of blindness in adults older than 55 years [
1]. With the increased ageing of society, it is estimated that the number of AMD patients will increase to 288 million in 2040 [
2]. There are two forms of AMD: wet or neovascular AMD and dry AMD. Although the wet form of AMD accounts for only 10 to 15% of cases, it is responsible for the majority of cases of severe vision loss [
3]. Neovascular AMD is characterized by choroidal neovascularization (CNV) in older people. Although effective benefits have been achieved with anti-VEGF treatment, choroidal neovascularization still progresses or regresses in some cases after 5 or more years of treatment [
1,
2,
4,
5]. Consequently, studies have show that blood inflammatory indices, angiogenesis-associated chemokine receptors such as IL-10, and CCR2
+ and CX3CR1
+ nonclassical monocytes were increased in refractory nAMD patients, suggesting that the poor therapeutic outcomes in the late stage may be related to macrophage overactivation and abnormal differentiation, but the specific mechanism is still unknown [
6,
7]. The precise mechanisms contributing to CNV continue to be elucidated.
In physiological and pathological conditions, the innate immune system plays a critical role in pathological angiogenesis, including endothelial cell proliferation, migration and vessel anastomosis [
8]. The links between macrophages and angiogenesis have been defined in several pathophysiological diseases, such as eye disorders, cancers with vigorous vessels and atherosclerosis [
9‐
11]. Macrophages have garnered increasing interest in the pathogenesis of AMD. Macrophages are a diverse group of cells originating from the mononuclear phagocytic lineage. They are highly plastic, exhibit dramatic phenotypic changes in response to various stimuli and exist in a multitude of subpopulations [
12]. Depending on the tissue context, macrophages can be classically (M1) or alternatively (M2) activated. Numerous studies have shown that macrophages that respond to injury in the early stage are an inflammatory phenotype (M1), and later, M1 cells shift towards an anti-inflammatory, proangiogenic, profibrotic, and wound healing phenotype (M2) [
13]. However, macrophages are highly plastic, and M1 macrophages can differentiate into the M2 phenotype in numerous diseases, such as chronic inflammatory diseases and cancers [
14]. Likewise, M2 macrophages can transition to M1 cells when treated with IFN-ɑ or antibodies against CD40 [
15], suggesting that the M1 and M2 macrophage phenotypes are reversible according to distinct immune environments. Studies on peripheral blood mononuclear cells from AMD patients showed that macrophage-derived miR-150 was significantly elevated, which was associated with pathologic angiogenesis in a VEGF-independent manner [
6]. Consistently, it was also demonstrated that the switch in macrophage phenotype from M1 to M2 was responsible for the increase in CNV in an old mouse model [
16]. A similar situation [
17] was observed in nAMD patients, in which alternatively activated ocular macrophages (M2) were the majority in the advanced stage of CNV [
18,
19]. Thus, examining dynamic changes in macrophage polarity could yield therapeutic outcomes in the treatment of neovascular diseases.
In our previous studies, the depletion of macrophages alleviated CNV progression in a mouse model, but the role of M1 and M2 macrophages was unclear. Furthermore, we inhibited M2 macrophages with a specific antibody, which alleviated CNV [
20]; however, the dynamic changes in macrophage differentiation are still unclear, which indicates a new basis for CNV treatment. In the present study, we used confocal immunofluorescence analysis to examine macrophage morphology in different CNV phases. Interestingly, we found that macrophages appeared immediately in response to laser injury in a mouse model and gradually infiltrated the CNV lesions. Then, the morphology of macrophages that initially appeared around the laser spot changed, and the morphology was distinct within or away from the vigorous neovascular area. With the growth of CNV, we found that macrophages gradually infiltrated the lesion and underwent morphological changes from branching to round-shaped. Furthermore, we identified these round-shaped macrophages as M2 macrophages that were restricted within or around the neovascular areas. Collectively, our data showed the dynamic characteristics of macrophage morphology in choroidal neovascularization, which may point to M2 polarity drift as an intervention target for the treatment of CNV. These data may also determine distinct immune environments around CNV lesions, which provides novel knowledge of macrophage polarization and critical treatment targets for CNV.