Resolvin D1 and lipoxin A4 improve alveolarization and normalize septal wall thickness in a neonatal murine model of hyperoxia-induced lung injury.

The critical fatty acids Docosahexaenoic Acid (DHA) and Arachidonic Acid (AA) decline in preterm infants within the first postnatal week and are associated with neonatal morbidities, including bronchopulmonary dysplasia (BPD). DHA and AA are precursors to downstream metabolites that terminate the inflammatory response. We hypothesized that treatment with Resolvin D1 and/or Lipoxin A4 would prevent lung injury in a murine model of BPD.To determine the effect of Resolvin D1 and/or Lipoxin A4 on hyperoxia-induced lung injury.C57/BL6 pups were randomized at birth to Room Air, Hyperoxia (>90% oxygen), Hyperoxia + Resolvin D1, Hyperoxia + Lipoxin A4, or Hyperoxia + Resolvin D1/Lipoxin A4. Resolvin D1 and/or Lipoxin A4 (2 ng/g) were given IP on days 0, 3, 6, and 9. On day 10, mice were sacrificed and lungs collected for morphometric analyses including Mean Linear Intercept (MLI), Radial Alveolar Count (RAC), and Septal Thickness (ST); RT-PCR analyses of biomarkers of lung development and inflammation; and ELISA for TGFβ1 and TGFβ2.The increased ST observed with hyperoxia exposure was normalized by both Resolvin D1 and Lipoxin A4; while, hyperoxia-induced alveolar simplification was attenuated by Lipoxin A4. Relative to hyperoxia, Resolvin D1 reduced the gene expression of CXCL2 (2.9 fold), TIMP1 (6.7 fold), and PPARγ (4.8 fold). Treatment with Lipoxin A4 also led to a reduction of CXCL2 (2.4 fold) while selectively increasing TGFβ2 (2.1 fold) and Smad3 (1.58 fold).The histologic and biochemical changes seen in hyperoxia-induced lung injury in this murine model can be reversed by the addition of DHA and AA fatty acid downstream metabolites that terminate the inflammatory pathways and modulate growth factors. These fatty acids or their metabolites may be novel therapies to prevent or treat lung injury in preterm infants

Biomarkers assayed by RT-PCR in lung tissue.

<p>TGF, transforming growth factor; VEGF, vascular endothelial growth factor, PPAR, peroxisome proliferator-activated receptor; BMP, bone morphogenetic protein; Eln, Elastin; Col1a1, Collagen, Type 1, alpha 1; TIMP1, TIMP metallopeptidase inhibitor 1; LOXL2, lysyl oxidase homolog 2; MMP, matrix metalloproteinase; CRP, C-reactive protein; IL, interleukin; CXCL2, chemokine (C-X-C motif) ligand 2; CD46, CD46 complement regulatory protein; ICAM-1, intracellular adhesion molecule 1; CCL5, Chemokine (C-C motif) ligand 5; TNF, tumor necrosis factor.</p

Fold change in gene expression within studied physiologic categories of lung development across experimental groups.

<p><b>Bold</b>  =  greater than a 2-fold change and/or p<0.05; values <1 represent down-regulation;</p><p>values >1 represent up-regulation.</p>a<p>Relative to the Room Air group;</p>b<p>Relative to the Hyperoxia alone group.</p><p>*p = 0.05,</p><p>**p<0.05.</p

Pup growth patterns by experimental group.

<p>RA, room air; H, hyperoxia; RvD1, Resolvin D1; LXA₄, Lipoxin A₄. *p<0.05 compared to Hyperoxia group.</p

Lung histology by treatment group.

<p>Representative images of H and E stained sections were taken at an original magnification of 200x. Bars represent 100 µm. RvD1, Resolvin D1; LXA₄, Lipoxin A₄.</p

Morphometric assessment of lung histology.

<p>Shown in Figure 3A, B, and C are the morphometric assessment of the mean linear intercept (MLI), radial alveolar count (RAC) and septal thickness (ST) respectively. Bars represent means +/- SEM. 37 mice were analyzed for the MLI and ST morphometric measures. A mean of 6 images were analyzed per section/mouse. 34 mice were analyzed for RAC. A mean of 3 images were analyzed per section per mouse. RA, room air; H, hyperoxia; RvD1, Resolvin D1; LXA₄, Lipoxin A₄ a = p<0.05 compared to RA group; b = p<0.05 compared to Hyperoxia alone group; c = p<0.05 compared to the Hyperoxia + RvD1 group.</p