Activation of immature microglia in response to stab wound in embryonic quail retina.

Activation of mature (ramified) microglia in response to injury in the adult central nervous system (CNS) is well documented. However, the response of immature (ameboid) microglia to injury in the developing CNS has received little attention. In this study, a stab wound was made in embryonic quail retinas at incubation days 7 and 9, and the response of retinal microglial cells was analyzed at different times between days 1 and 37 postinjury. The appearance of microglial cells within the wound occurred at the same time as the arrival of the first migrating ameboid microglial cells at an equivalent area in control retinas. Therefore, no specific attraction of microglia toward the wound was observed. Microglial cells in the wound had phenotypic features similar to those of activated microglia in the adult CNS. Thus, their shape was more compact compared with microglial cells outside the wound, expression of the molecule recognized by the QH1 antibody was up-regulated, and their lysosomal compartment was markedly increased. Transitional forms between normal ameboid and activated-like microglial cells were seen at the wound edge, supporting the view that ameboid microglia become activated when they contact the wound during the normal course of their migration in the retina. The microglial reaction was maintained within the wound at 37 days postinjury. In addition to the stab wound, secondary damage areas were found in experimental retinas. Activated cells could still be observed in these areas at 37 days postinjury.

Behavior of in vitro cultured ameboid microglial cells migrating on Müller cell end-feet in the quail embryo retina.

Ameboid microglial cells migrate tangentially on the vitreal part of quail embryo retinas by crawling on Müller cell end-feet (MCEF) to which they adhere. These microglial cells can be cultured immediately after dissection of the eye and isolation of sheets containing the inner limiting membrane (ILM) covered by a carpet of MCEF (ILM/MCEF sheets), to which the cells remain adhered. Morphological changes of microglial cells cultured on ILM/MCEF sheets for 4 days were characterized in this study. During the first minutes in vitro, lamellipodia-bearing bipolar microglial cells became rounded in shape. From 1 to 24 h in vitro (hiv), microglial cells swept and phagocytosed the MCEF on which they were initially adhered, becoming directly adhered on the ILM. MCEF sweep was dependent on active cell motility, as shown by inhibition of sweep after cytochalasin D treatment. From 24 hiv on, after MCEF phagocytosis, microglial cells became more flattened, increasing the surface area of their adhesion to substrate, and expressed the beta1 subunit of integrins on their membrane. Morphological evidence suggested that microglial cells migrated for short distances on ILM/MCEF sheets, leaving tracks produced by their strong adhesion to the substrate. The simplicity of the isolation method, the immediate availability of cultured microglial cells, and the presence of multiple functional processes (phagocytosis, migration, upregulation of surface molecules, etc.) make cultures of microglial cells on ILM/MCEF sheets a valuable model system for in vitro experimental investigation of microglial cell functions.