Nitric oxide counteracts the hyperoxia-induced proliferation and proinflammatory responses of mouse astrocytes

Abstract

Preclinical studies in the premature baboon evaluating the efficacy and potential toxicity of inhaled nitric oxide indicated a significant effect on astrocyte area density, suggesting phenotypic and functional changes in astrocytes upon exposure to nitric oxide. However, the effects of nitric oxide and oxygen, the two major therapeutic gases utilized in neonatal intensive care, on astrocyte morphology and function remain vastly unknown. Herein, we report that exposure of mouse neonatal cortical astrocytes to hyperoxia results in a proinflammatory phenotype and increase in proliferation without significant changes in cellular morphology or levels of intermediate filament proteins. The proinflammatory phenotype was evident by a significant increase in cellular levels of cyclooxygenase-2 and a concomitant increase in prostaglandin E(2) secretion, a decline in the intracellular and secreted levels of apolipoprotein E, and a significant increase in the intracellular levels of clusterin. This proinflammatory phenotype was not evident upon simultaneous exposure to hyperoxia and nitric oxide. These results suggest that exposure to nitric oxide in the setting of hyperoxia confers unrecognized beneficial effects by suppressing astrocytic inflammation.

Figure 1

Panel A: Representative images of astrocytes in culture after staining with anti-GFAP antibody (green) following exposure to hyperoxia, nitric oxide and hyperoxia plus nitric oxide (blue, DAPI nuclear stain). Panel B: representative western blots of GFAP evaluating the levels of GFAP in astrocytes after the different exposures. Panels C, D and E: Levels of GFAP, vimentin and proliferation marker PCNA quantified by densitometric analysis of western blots and normalized to GAPDH levels, respectively. Data is expressed as fold change (mean ± SEM) from the corresponding controls, n=3 for all exposure groups and controls. Open box, control cells; black box, hyperoxia exposed cells; grey box, nitric oxide exposed cells and hatched box, nitric oxide plus hyperoxia exposed cells. *p<0.05 compared to the corresponding room air exposed astrocytes.

Figure 2

Panel A: Cellular COX-2 levels normalized to GAPDH and expressed as fold change from the corresponding control, n=3 for all exposure groups; values represent mean ± SEM. Panel B: Levels of PGE₂ in the astrocyte conditioned media *p<0.05 compared to the corresponding room air exposed astrocytes, n=3 for all exposure groups. Open box, control cells; black box, hyperoxia exposed cells; grey box, nitric oxide exposed cells and hatched box, nitric oxide plus hyperoxia exposed cells.

Figure 3

Panel A and B: Intracellular and extracellular levels of ApoE. Data is expressed as fold change from the corresponding control after normalizing to GAPDH, for the intracellular levels and to total protein for the extracellular levels *p<0.05 compared to the corresponding room air exposed astrocytes, n=3 for all exposure groups; values represent mean ± SEM. Open box, control cells; black box, hyperoxia exposed cells; grey box, nitric oxide exposed cells and hatched box, nitric oxide plus hyperoxia exposed cells. Insert Panel A and B: Representative western blots using anti-ApoE antibody. Since each experimental condition had a corresponding control the western blots were performed in pairs in different gels. Lanes 1 and 2 control and hyperoxia exposed cells respectively; Lanes 3 and 4, control and nitric oxide exposed cells; and Lanes 5 and 6 cells, control and cells exposed to nitric oxide plus hyperoxia.

Figure 4

Panels A and B: Intracellular and extracellular levels of clusterin. Data is expressed as fold change from the corresponding control after normalizing to GAPDH, for the intracellular levels and to total protein for the extracellular levels *p<0.05 compared to the corresponding room air exposed astrocytes, n=3 for all exposure groups; values represent mean ± SEM. Open box, control cells; black box, hyperoxia exposed cells; grey box, nitric oxide exposed cells and hatched box, nitric oxide plus hyperoxia exposed cells.