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A Nestler, J Kacza, P Wiedemann, J Seeger; Phagocytosis-dependent and Immunocytochemical Characterization of Microglia in the Axotomized Rat Retina . Invest. Ophthalmol. Vis. Sci. 2002;43(13):759.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To characterize interactions between activated microglia and degenerating retinal ganglion cells using a combined phagocytosis-dependent and immunocytochemical labelling technique. Methods:Adult Wistar rats received an unilateral intraorbital transsection of the optic nerve followed by deposition of the membranophilic fluorescent dye 4Di-10ASP onto the ocular nerve stump in order to retrogradely label the retinal ganglion cells (RGCs). After survival times of two days up to one year the retinae were flat-mounted, labelled cells were photoconverted using a closed conversion chamber. Additionally, activated microglia were immunocytochemically marked using the monoclonal antibody OX-42 that recognizes the CR3 complement receptor. Fluorescence, light and transmission electron microscopy were performed. Results:Activated microglial cells got transcellularly labelled by phagocytosing the debris of degenerating RGCs. OX-42 immunocytochemistry led to a fine and demarcated staining of the microglial cell membrane revealing the morphological complexity of the microglial cells typical ramifications. This was very useful to identify numbers of complex shaped filopodia-like processes, which were found to enwrap heavily degraded RGCs. The incorporation of small fractions of membranous material into microglial cells was observed, whereas the phagocytosis of an entire RGC body was not found. Ultrastructural analysis confirmed that phagosomes were located in microglia but not in Müller cells or astrocytes. Conclusion:The applied double labelling technique turned out to be a versatile tool for a simultaneous demonstration of different stages and ways of cellular interaction of phagocytic microglia in the injured central nervous system.
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