The Relationship of Microglial Cells to Dying Neurons During Natural Neuronal Cell Death and Axotomy-induced Degeneration of the Rat Retina.

Abstract

The interactions between dying neurons and phagocytotic cells within the developing and injured retina remain controversial. The present work explored the role of microglia and investigated whether so-called resident microglial cells are permanently responsible for removing cell debris whenever it is produced. As a first goal, I characterized some quantitative and morphometric features of the small ipsilateral retinocollicular projections and analysed the permanent function of phagocytosing microglia with these projections as a paradigm. To achieve this, I combined the fluorescent dyes Dil and 4Di-10ASP, both of which persist in the labelled ganglion cells after injection into the superior colliculus (SC), and retrograde labelling. After neuronal degradation, the dyes accompany the degradation products, become interiorized and then persist within the phagocytosing microglia. Consequently, early labelling of microglial cells can be assessed by injecting one dye into the SC during the first postnatal day of life, that is, prior to advanced natural neuronal cell death. Labelling of the remaining ipsilaterally projecting neurons with the second dye following intraorbital axotomy in adulthood and during subsequent neuronal death would therefore result in double labelling of some microglial cells, if these were involved in phagocytosis during both the natural and the induced phases of neuronal degradation. The ganglion cells which survived natural neuronal cell death remained fluorescent for 3 months after labelling with either dye on the day of the animal's birth, indicating that both fluorescent probes persisted within neurons. Quantitatively, 1770+/-220 ganglion cells/mm2 were labelled within the contralateral retina and a total population of 1442+/-120 cells/retina were observed within the periphery of the inferior/temporal quadrant of the ipsilateral retina. A smaller, ipsilateral projection of 150+/-24 cells/retina was uniformly scattered throughout the rest of the retinal surface. Transient projections of ganglion cells to either the contralateral or the ipsilateral colliculi and death of labelled ganglion cells during the first postnatal days resulted in labelling of 210+/-36 microglial cells/mm2 within the contralateral retina and a total number of 800+/-120 cells/retina within the inferior/temporal and 200+/-22 cells/retina within the rest of the retina. These labelled microglial cells were observed in adulthood and indicated that after taking away the neuronal cell debris they persisted within the retinal tissue. The small number of prelabelled ganglion cells which formed persistent ipsilateral projections until adulthood were axotomized by transecting the optic nerve, and resulted in additional labelling of microglial cells with the second fluorescent dye as well. Double-labelled microglia were observed within the inferior/temporal quadrant (3500+/-240 cells/retina) and to a lesser extent (340+/-40 cells/retina) scattered over the entire retinal surface. The chronotopological sequence of microglial labelling paralleled that of ganglion cell degeneration. Injection of protease inhibitors into the vitreous body during optic nerve transection retarded retrograde glial cell degeneration, probably by blocking microglial proteases. The results directly proved that the same microglial cells which remove neuronal cell debris in the postnatal retina were reactivated later in life to proteolytically degrade and then phagocytose neurons which had altered because of the axotomy.