LPS-stimulated microglial cells promote ganglion cell death in organotypic cultures of quail embryo retina.

During development microglia colonize the central nervous system (CNS) and play an important role in programmed cell death, not only because of their ability to remove dead cells by phagocytosis, but also because they can promote the death of neuronal and glial cells. To study this process, we used as experimental systems the developing in situ quail embryo retina and organotypic cultures of quail embryo retina explants (QEREs). In both systems, immature microglia show an upregulation of certain inflammatory markers, e.g., inducible NO synthase (iNOS), and nitric oxide (NO) under basal conditions, which can be further enhanced with LPS-treatment. Hence, we investigated in the present study the role of microglia in promoting ganglion cell death during retinal development in QEREs. Results showed that LPS-stimulation of microglia in QEREs increases (i) the percentage of retinal cells with externalized phosphatidylserine, (ii) the frequency of phagocytic contacts between microglial and caspase-3-positive ganglion cells, (iii) cell death in the ganglion cell layer, and (iv) microglial production of reactive oxygen/nitrogen species, such as NO. Furthermore, iNOS inhibition by L-NMMA decreases cell death of ganglion cells and increases the number of ganglion cells in LPS-treated QEREs. These data demonstrate that LPS-stimulated microglia induce ganglion cell death in cultured QEREs by a NO-dependent mechanism. The fact that phagocytic contacts between microglial and caspase-3-positive ganglion cells increase suggests that this cell death might be mediated by microglial engulfment, although a phagocytosis-independent mechanism cannot be excluded.

Onset of microglial entry into developing quail retina coincides with increased expression of active caspase-3 and is mediated by extracellular ATP and UDP.

Microglial cell precursors located in the area of the base of the pecten and the optic nerve head (BP/ONH) start to enter the retina of quail embryos at the 7th day of incubation (E7), subsequently colonizing the entire retina by central-to-peripheral tangential migration, as previously shown by our group. The present study demonstrates a precise chronological coincidence of the onset of microglial cell entry into the retina with a striking increase in death of retinal cells, as revealed by their active caspase-3 expression and TUNEL staining, in regions dorsal to the BP/ONH area, suggesting that dying retinal cells would contribute to the microglial cell inflow into the retina. However, the molecular mechanisms involved in this inflow are currently unclear. Extracellular nucleotides, such as ATP and UDP, have previously been shown to favor migration of microglia towards brain injuries because they are released by apoptotic cells and stimulate both chemotaxis and chemokinesis in microglial cells via signaling through purinergic receptors. Hence, we tested here the hypothesis that ATP and UDP play a role in the entry and migration of microglial precursors into the developing retina. For this purpose, we used an experimental model system based on organotypic cultures of E6.5 quail embryo retina explants, which mimics the entry and migration of microglial precursors in the in situ developing retina. Inhibition of purinergic signaling by treating retina explants with either apyrase, a nucleotide-hydrolyzing enzyme, or suramin, a broad spectrum antagonist of purinergic receptors, significantly prevents the entry of microglial cells into the retina. In addition, treatment of retina explants with either exogenous ATP or UDP results in significantly increased numbers of microglial cells entering the retina. In light of these findings, we conclude that purinergic signaling by extracellular ATP and UDP is necessary for the entry and migration of microglial cells into the embryonic retina by inducing chemokinesis in these cells.

Microglial Activation Promotes Cell Survival in Organotypic Cultures of Postnatal Mouse Retinal Explants.

The role of microglia during neurodegeneration remains controversial. We investigated whether microglial cells have a neurotoxic or neuroprotective function in the retina. Retinal explants from 10-day-old mice were treated in vitro with minocycline to inhibit microglial activation, with LPS to increase microglial activation, or with liposomes loaded with clodronate (Lip-Clo) to deplete microglial cells. Flow cytometry was used to assess the viability of retinal cells in the explants and the TUNEL method to show the distribution of dead cells. The immunophenotypic and morphological features of microglia and their distribution were analyzed with flow cytometry and immunocytochemistry. Treatment of retinal explants with minocycline reduced microglial activation and simultaneously significantly decreased cell viability and increased the presence of TUNEL-labeled cell profiles. This treatment also prevented the migration of microglial cells towards the outer nuclear layer, where cell death was most abundant. The LPS treatment increased microglial activation but had no effect on cell viability or microglial distribution. Finally, partial microglial removal with Lip-Clo diminished the cell viability in the retinal explants, showing a similar effect to that of minocycline. Hence, cell viability is diminished in retinal explants cultured in vitro when microglial cells are removed or their activation is inhibited, indicating a neurotrophic role for microglia in this system.