Jyoti Watters, Stephanie Smith, Gordon Mitchell, Scott Friedle, Christine Sibigtroth, Stéphane Vinit

Hypoxia and increased extracellular nucleotides are frequently coincident in the brainstem. Extracellular nucleotides are potent modulators of microglial inflammatory gene expression via P2X purinergic receptor activation. Although hypoxia is also known to modulate inflammatory gene expression, little is known about how hypoxia or P2X receptor activation alone affect inflammatory molecule production in brainstem microglia, nor how hypoxia and P2X receptor signaling interact when they occur together. In this study, we investigated the ability of a brief episode of hypoxia (2hrs) in the presence and absence of the non-selective P2X receptor agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) to promote inflammatory gene expression in brainstem microglia in adult rats. We evaluated inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) mRNA levels in immunomagnetically-isolated brainstem microglia. Whereas iNOS and IL-6 gene expression increased with hypoxia and BzATP alone, TNFα expression was unaffected. Surprisingly, BzATP-induced inflammatory effects are lost after hypoxia, suggesting that hypoxia impairs pro-inflammatory P2X receptor signaling. We also evaluated the expression of key P2X receptors activated by BzATP, namely P2X1, P2X4 and P2X7 receptors. Whereas hypoxia did not alter their expression, BzATP upregulated P2X4 and P2X7 mRNAs; these effects were ablated in hypoxia. Although both P2X4 and P2X7 receptor expression correlated with increased microglial iNOS and IL-6 levels in microglia from normoxic rats, in hypoxia, P2X7 only correlated with IL-6, and P2X4 correlated only with iNOS. In addition, correlations between P2X7 and P2X4 were lost following hypoxia, suggesting that P2X4 and P2X7 receptor signaling differs in normoxia and hypoxia. Together, these data suggest that hypoxia suppresses P2X receptor-induced inflammatory gene expression, indicating a potentially immunosuppressive role of extracellular nucleotides in brainstem microglia following exposure to hypoxia.