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Updated: Aug 20, 2026
Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes
Published on: June 17, 2020
A novel rat model to study the functions of macrophages during normal development and pathophysiology of the eye
Stacey Hose1, J Samuel Zigler, Debasish Sinha
1Department of Ophthalmology, School of Medicine, The Johns Hopkins University, 600 North Wolfe Street/Jefferson 3-127A, Baltimore, MD 21287, USA.
Abstract:
Several studies have shown that macrophages play an active role in the initiation and completion of the programmed cell death process during development. Macrophages are called professional phagocytes, as their primary role is phagocytosis. The process of phagocytosis is complex and to date only poorly defined. It has also been postulated that macrophages around the developing lens likely migrate into the neural retina and differentiate into microglia after completion of their role as debris removers. We have identified ED1 immunopositive macrophages and CD11b/18 (OX-42) immunopositive macrophage-like cells in the vitreous chamber and sub-retinal space of a rat spontaneous mutation that we have termed Nuc1. The mutation appears to affect the programmed cell death process and is highly eye specific in its effects. While ED1 and ED2-immunopositive macrophages have previously been found surrounding the developing lens and are thought to play a role in the programmed regression of the tunica vasculosa lentis (part of the vascular structure present on the posterior surface of the lens during development), OX-42-immunopositive cells have not previously been identified in the vitreous chamber under normal or pathological conditions. Macrophage subpopulations surrounding the lens may differentiate into OX-42+ cells in Nuc1 following the release of lens material into the vitreous after the posterior capsule ruptures. In Nuc1 homozygotes, the posterior lens capsule ruptures before birth, causing lens material to be extruded into the vitreous compartment and damaging the tunica vasculosa lentis. Alternatively, OX-42+ cells may be recruited due to an inflammatory response both in the vitreous compartment and sub-retinal space. Inflammation is known to have an enhanced influx of phagocytic cells. Our data suggests that subpopulations of macrophages perform distinct functions in inducing apoptosis and phagocytic activity during normal conditions and in disease.
Insights
Macrophages play a key role in programmed cell death and debris removal during development. In the Nuc1 rat mutation, distinct macrophage subpopulations exhibit unique functions in apoptosis and phagocytosis, particularly in eye-specific developmental defects.
Area of Science:
- Immunology
- Developmental Biology
- Ophthalmology
Background:
- Macrophages are professional phagocytes crucial for programmed cell death and debris removal during development.
- Their role in complex phagocytosis processes and potential differentiation into microglia requires further definition.
- Previous studies identified ED1 and ED2 macrophages around the developing lens, involved in tunica vasculosa lentis regression.
Purpose of the Study:
- To investigate the role and characteristics of macrophage subpopulations in the Nuc1 rat mutation, which affects programmed cell death.
- To identify and characterize OX-42 immunopositive macrophage-like cells in the vitreous and sub-retinal space of Nuc1 rats.
- To explore the functional distinctiveness of macrophage subpopulations in apoptosis and phagocytosis during normal development and disease.
Main Methods:
- Identification of ED1 and OX-42 immunopositive cells in the vitreous chamber and sub-retinal space of Nuc1 rats.
- Analysis of the Nuc1 mutation's eye-specific effects on programmed cell death and lens development.
- Comparison of macrophage populations in Nuc1 mutants with normal conditions.
Main Results:
- ED1 immunopositive macrophages and OX-42 immunopositive macrophage-like cells were identified in the vitreous and sub-retinal space of Nuc1 rats.
- The Nuc1 mutation causes posterior lens capsule rupture before birth, leading to lens material extrusion and damage to the tunica vasculosa lentis.
- OX-42+ cells were found in the vitreous chamber, a novel finding under normal or pathological conditions, potentially differentiating from lens-associated macrophages or recruited by inflammation.
Conclusions:
- Subpopulations of macrophages perform distinct functions in apoptosis and phagocytosis during normal development and in the Nuc1 mutation.
- The Nuc1 mutation provides a model to study macrophage differentiation and function in response to lens injury and inflammation.
- Further research is needed to fully elucidate the complex roles of macrophages in ocular development and disease.
