Interplay of inflammation and hypoxia in perinatal brain injury

Aims: Degree of perinatal brain damage due to hypoxia depends on duration and severity of the insult as well as on the stage of brain maturation. Additionally, inflammatory pathways play a critical role. Endotoxins like LPS sensitize the immature brain to a later hypoxic-ischemic injury. Here, we investigated the impact of microglia in neuroinflammation as well as the interplay of different stimuli in microglia activation in vitro.

Methods: BV2 cells were treated for 24 hours with 0 – 100 µg/mL LPS before exposition to 1% O2 for 6 hours. Expression of key players of pathways regulated by hypoxia and/or inflammation such as HIF-1α, NFκB, iNOS, TNFα, and IL1β were analyzed by quantitative real-time polymerized chain reaction, Western Blot, and ELISA. Nitric oxide production was quantified by the Griess assay.

Results: Exposition of BV2 cells to hypoxia increased the expression of HIF-1α and iNOS mRNA within 24-hour poststimulation (two- and threefold, respectively) as well as expression of NFκB (sixfold), TNFα (twofold), and IL1β mRNA levels (fivefold) within 48 hours. In contrast, a rapid increase of TNFα (14-fold) and iNOS (129-fold) mRNA could be observed 3 and 6 hours post-LPS stimulation, respectively. Thereby, NFκB and IL1β expression peaked at 12 and 24 hour post-LPS exposition (2000 and 2100 fold). Surprisingly, LPS stimulation mediated also an upregulation of the hypoxia inducible factor HIF-1α (5-fold) in BV2 cells suggesting a synergistic regulation of hypoxia- and inflammation-induced transcriptional pathways in microglia. Notably, nitric oxide secretion as a result of iNOS mRNA over expression in LPS/hypoxia-activated BV2 cells was up to 52% higher than the accumulated effects of LPS and hypoxia alone.

Conclusion: Present data indicate that LPS exposition and hypoxia induce additive effects in microglia. Thereby, LPS-stimulated inflammation mediates a long lasting sensitization of BV2 cells to hypoxia. Nitric oxide toxicity mediated by activated microglia may contribute to delayed neural damage following cerebral hypoxia.