Introduction.At the present stage, the development of the human eye has been studied to a greater extent on animal models [1, 2, 3]. The problems of congenital pathologies of the human eye are one of the main aspects studied in modern most relevant research. One of the little-studied issues of eye development is the study of the mechanisms of retinal development, the participation of macrophages in the formation of the vascular system of the eye, migration of neurons and glia in the formation of retinal layers. The upper and lower eyelids merge during development and then separate again. The mechanism for re-detaching the eyelids is still unclear. These questions determined the direction of our research, since they can contribute to solving the problem of angiogenesis and vascular involution [4, 5, 6].
Choroidal neovascularization (CNV) directly related to the loss of vision in certain eye diseases such as age-related macular degeneration. Although several histological studies in humans have confirmed the involvement of macrophages in the formation of CNV, the exact mechanisms are still not fully understood, the submission can only be based on a consideration of this process in human ontogenesis [7, 8, 9]. Comprehensive insights can enable the control of key targets in these complex eye development processes. Microglia are cells of non-neuronal origin that are situated in the central nervous system. Early macrophages of zebrafish migrate from the yolk sac to the brain and retina in 26-30 hours post fertilization (hpf) and transform into microglia in 55-60 hpf. Migration of macrophages into the central nervous system requires signaling from the factor-1 receptor that stimulates macrophage colonies (csf-1r), which is encoded by the gene fms [10, 11, 12]. It was found that targeted knockdown of csf-1r morpholino oligonucleotides delays the migration of macrophages from the yolk sac to the retina, and this delay migration of macrophages leads to microphthalmia, output delay of the cell cycle among retinal progenitors and the absence of neuronal differentiation [13, 14, 15]. If embryos survived under conditions of morpho-dependent inhibition, translation was lost, and microglia migrated repeatedly to the retina and neuronal differentiation was partially restored. However, it is not known whether microglial migrants had phagocytic activity, which requires further deeper research.
Purpose of our research is obtaining new data on the role of macrophages in the morphogenesis of the human retina, contributing to the development of pathogenetically based strategies in the prevention and treatment of disorders of the development of the organ of vision.
Material and methods. The study was performed taking into account the provisions of the Helsinki Declaration (2000, 2013) and with the permission of the ethical committee of the Far Eastern Federal University. The localization of CD163 positive cells in the dynamics of development of the structures of the human eye was revealed using the material of 15 human embryonic and fetal eyes by the method of immune histochemistry. Stained sections of biopsy specimens were performed according to classical protocol for a method using hematoxylin and eosin.
Results of analysis of literature data. It is known that the only ocular field is formed in the center of the front of the neural plate during gastrulation; it is characterized at the molecular level the expression "eye field transcription factors". The single field of the eye is divided into two, forming the optic vesicle, and then (under the influence of the lens placode) the optic cup. The lens develops from the lens placode (superficial ectoderm) under the influence of the underlying optic bubble. PAX6 acts as a major control gene in this phase and activates genes encoding cytoskeletal proteins, structural proteins, or membrane proteins. It is known that the endothelial growth factor is affiliated with PAX1, which is responsible for the axial organization of the human embryo organism; therefore, it is functionally linked directly to macrophages that secrete VEGF. The cornea forms from the superficial ectoderm, and cells from the periocular mesenchyme migrate into the cornea, giving rise to the future corneal stroma. In the same way, the iris and ciliary body are formed from the optic cup. The outer layer of the optic cup becomes pigmented retinal epithelium, while the bulk of the inner layer of the optic cup later forms the neural retina with six different cell types, including photoreceptors. Although fish, unlike higher vertebrates, are capable of growth throughout their life, the environment is also an important factor in controlling the development of eyes in this group, and eye development is not strictly genetically determined.
Many structures in the developing eye are temporary and undergo involution during development. The apoptosis of cells of these structures and their phagocytosis are triggered with the participation of macrophages. Tunica vasculosa lentis (TVL) is a temporary vasculature surrounding the developing lens that regresses prenatally in humans and is a prime example of regulated involution.Research has shown that macrophages play an active role in initiating and completing the process of programmed cell death during human eye development. Macrophages are called professional phagocytes because their main role is phagocytosis. The process of phagocytosis in the structures of the human eye is complex and poorly understood today. It has also been suggested that macrophages around the developing lens are likely to migrate into the neural retina and differentiate into microglia after completing their role as removal (fig.1).
Figure 1 - Macrophages in the developing vitreous humor of a human fetus for 10 weeks. Immune histochemistry for the detection of CD68 positive cells. Phagocytosis of children of the mesenchymal vitreous body. Magnification x400.
The retinal ganglion cells grow in the direction of the optic nerve to form the optic tract. Cellular processes during eye development known in frogs, zebrafish, chickens, and mice, as well as established differences between species, represent directions in the study of missing links for future research. It was found that macrophages expressing CD163, located in the ectomesenchyme surrounding the optic vesicle, were detected in the head section of the human embryo from the end of the 3rd week. The presence of macrophages with expression of CD163 is due to the fact that the development of human eye structures depends on the main induction mechanism of differentiation, which is realized through the interaction of macrophages and cells of the forming eye vesicle. Macrophages secrete TGF-beta, are able to phagocytose IgG-associated latent TGF-beta complexes, and release active TGF-beta into the extracellular matrix to induce apoptosis of various cell types through signaling pathways: SMAD and DAXX. TGF-beta activation depends on various factors that activate signaling pathways. We noted that in areas with high proliferative activity, cells with the CD163 phenotype are absent, earlier being detected in the inner layers of the retina, which indirectly confirms their main role at this stage in the supply of signaling molecules specifically for differentiating cells, for the appearance of various repression of cell genomes and directed induction of cell differentiation in various tissues of the human eye.
Both apoptotic cells and macrophages are identified in the area of the eyelid junction and during cell proliferation. In the tissues of the choroid, in the formation zone of the anterior chamber of the eye, TUNEL-positive cells and immunohistochemically positive macrophages were also found at this stage. This suggests that apoptosis, like phagocytosis, can also play an important role in providing space for the proliferation of newly organized epidermal cells without wide intercellular spaces. In addition, it is recognized that the progressive differentiation of epithelial cells from the skin of the eyelids to the connective region of the conjunctiva may also play a role in eyelid separation (fig. 2).
Figure 2 - Apoptosis of the eye structures of a human fetus at 14 weeks. Vascular involution. Immune histochemistry to identify cells expressing CD68. Magnification x400.
The data of our study expand the diagnostic resource for identifying the causes of congenital retinal pathology in premature infants and are necessary to create a fundamental platform in the development of new, more effective and pathogenetically determined conservative methods of treatment and prevention of eye pathology in newborns.
Conclusion.Microglial cells / macrophages are essential for normal retinal growth and neurogenesis. This study provides new insights into the neurogenic role of microglia during human retinal development.
Macrophages secreting vascular endothelial growth factor (VEGF) regulate angiogenesis in the structures of the human eye during ontogenesis.
Inflammation of the organ of vision is associated with an increased influx of phagocytic cells. According to our data, it can be assumed that subpopulations of macrophages perform different functions in the induction of apoptosis and phagocytic activity in physiological regeneration and under conditions of pathological development of the organ of vision.
The study was financially supported by the International Medical Research and Education Center (Vladivostok, Russia)
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