Extracellular Adenosine Protects Microglia-like Cells During Simulated Ischemia

Microglia are the surveillant immune cells of the brain, and as such they help protect the nervous system by clearing pathogens and dead cells, producing neurotrophic factors, and maintaining homeostasis. As guardians of the brain, microglia may be especially important in neural repair following injuries such as ischemic stroke, a disease which takes 6.2 million lives worldwide every year. Although microglia respond to tissue injury after stroke, they are themselves also vulnerable to ischemic death. Protecting microglia from ischemia-induced cell death could promote brain tissue restoration after stroke. Given the well-known anti-inflammatory and protective effects of adenosine (Ado) on other cell types following ischemia, we investigated the effects of Ado on MG during ischemia. Here, we provide preliminary evidence in an in-vitro model that exogenous Ado can attenuate MG cell death during stroke. Using cultured microglia-like BV2 cells in oxygen-glucose deprivation (OGD) conditions to mimic ischemic stroke, we found that application of 500 µM Ado during OGD significantly (p=.00002) reduced cell death (Cell Death Ratios- Control: 0.12±0.03, OGD: 0.23±0.08, OGD+500µM Ado: 0.08±0.03). Preliminary evidence also indicates that Ado (500 µM) protects parenchymal microglia against OGD in a neonatal mouse hippocampal slice model. We are currently investigating the receptor(s) (R) responsible for mediating the protection. Preliminary studies using 35 nM 2-Chloro-N6-cyclopentyladenosine (an A1-R agonist) and 200 nM CGS21680 (an A2A-R agonist) indicate that neither A1-R nor A2A-R alone are sufficient to cause the protective effects of Ado (p=0.70 and p=0.34 respectively). Using 2 nM ZM 241385 (an A2A-R antagonist) was also not able to block the protective effects of adenosine, indicating that the A2A-R is not necessary for this protective effect. To continue these experiments, we have begun to extend our project into a line of primary microglial cells, to create a model that is even closer to resident microglia during stroke. Moving forward, using both Ado-R agonists and antagonists to identify the specific Ado-R(s) protecting microglia during stroke would not only suggest a new role for adenosine in microglial signaling, but could also give rise to a new pharmaceutical target for either inducing or preventing microglial cell death during ischemia.