Apoptotic cell death and microglial cell responses in cultured rat retina

Background. Optic nerve transection results in degeneration of axotomized retinal ganglion cells followed by the activation of resident microglial cells. Methods. An organotypic culture of neonatal rat retina was used to examine the temporal aspect of retinal ganglion cell death and microglial cell recruitment. Retinas were fixed at various times after explantation and prepared for immunohistochemistry and lectin staining. Results. Terminal deoxytransferase dUTP nick-end labeling (TUNEL) and immunohistochemistry for cleaved caspase-3 demonstrated a massive cascade of cell death in the ganglion cell layer (GCL) within hours after explantation. The rate of cell death in this layer was high and continued over a period of 48 h. In contrast, the rate of cell death was low in the outer nuclear layer (ONL) and apoptotic cells were evident after 6 days in vitro. Increases in the density of microglial cells in the GCL appeared to be recruited by proliferation within hours after explantation. In parallel, resident microglial cells also acquired an activated morphology as revealed by isolectin B-4 staining. Microglial cell activation in the GCL also included an upregulated expression for the lysosomal protein ED-1 and the cysteine protease inhibitor cystatin C. After 1 week of culture, immunolabeling for ED-1 demonstrated the presence of activated microglial cells also in the ONL. Conclusion. These data show rapid microglial cell recruitment and activation following the axotomy-induced cell death of differentiated ganglion cells. The processes of microglial cell activation and cell death are slower in the outer retina.