Purinergic regulation of microglial mobility and viability during ischemic conditions in developing mouse brain tissues

Microglia (MG) are the resident macrophages of the central nervous system (CNS) and perform both supportive and immune functions. Although it was traditionally thought that MG exist in an inactive state until some form of tissue injury or pathology, recent imaging studies have provided abundant evidence that MG in the uninjured adult brain and spinal cord are not morphologically silent. However, the dynamic behavior and function of MG in the developing brain normally as well as during pathology has been less studied. Here, we investigated MG behavior in freshly prepared hippocampal slices from neonatal transgenic mice during the first week of birth. The results are presented in three data chapters including: (1) a description of early MG mobilization during the first postnatal week; (2) a description of the effects of simulated ischemia on MG mobilization and viability during the first postnatal week and (3) a presentation of evidence for the regulation of ischemia-induced MG cell death by the activation of the P2X7 receptor.

In the first data chapter, a detailed study of MG ramification, density, and mobilization during the first postnatal week in the developing hippocampus was performed. With regards to MG mobilization, we distinguished between MG migration (involving cell soma translocation) and motility (involving cell process changes). The results show an initial increase followed by a decline in mobilization of hippocampal MG during the first postnatal week. MG mobilization to neuronal cell body rich regions was hampered in apoptosis-deficient animals suggesting that apoptotic factors regulate MG mobilization in vivo.

In the second data chapter, neonatal slices were subjected to ischemia, simulated by oxygen and glucose deprivation (OGD). Both transient (2hr) and prolonged (6hr) OGD resulted in decreased MG mobilization and viability, though MG from younger tissues were less sensitive than older ones to OGD. In the final data chapter, the mechanism of MG cell death induced by OGD was investigated. MG cell death during OGD is shown to be dependent on both calcium signaling and extracellular purines. Specifically, using both genetic and pharmacological approaches, inhibiting P2X7 receptor signaling is shown to reduce MG cell death. Finally, data is presented that suggest that this cell death may occur in a cell autonomous manner since MG express functional P2X7 receptors and OGD induces cell death in a MG cell line that is reduced by P2X7 receptor antagonism.

The results presented herein provide novel insights into MG behavior during development under normal and ischemic conditions providing the first evidence for rapid changes in MG mobilization in the developing mouse brain and P2X7 regulated MG cell death during ischemia. With our results, we extend the concept of MG as patrolling cells in the developing brain (in distinction from surveying cells in the adult brain) to rodents and suggest targeting the P2X7 receptor to enhance MG survival as a strategy for stroke therapy.