Hyaluronan content in bronchoalveolar lavage and serum, and expression of enzymes regulating hyaluronan synthesis and degradation.

<p>HA content was determined in BAL (A) and in serum (B). At the end of asphyxia, the HA content of BAL decreased and that in the serum increased. Resuscitation with 21% O₂ did not increase HA either in the BAL or in the serum. However, BAL HA was significantly increased in animals resuscitated with 100% O₂, and this increase was completely inhibited by treatment with NAC (A). The changes in hyaluronan synthase 1 (<i>has1</i>), <i>has2</i> and <i>has3</i>, as well as hyaluronidase 1 (<i>hyal1</i>) and 2 (<i>hyal2</i>) were determined using quantitative RT-PCR. There were no changes in <i>has1</i> and <i>has 3</i> expression (data not shown). Expression of <i>has2</i> (C) showed a trend to increased expression after asphyxia and 21% O₂ resuscitation. However, treatment with NAC significantly inhibited has2 expression compared to 100% O₂ resuscitation alone (C). The expression of <i>hyal1</i> (D) and <i>hyal2</i> (E) remained unchanged except that 100% O₂ exposed animals treated with NAC showed significantly increased expression of both hyaluronidases.</p

Lung myeloperoxidase and N-acetylglucosaminidase activities as measures of neutrophil and macrophage contents respectively.

<p>Macrophage content was determined by NAG activity and neutrophil content was determined by MPO activity as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038839#pone.0038839-Savani4" target="_blank">[82]</a> and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038839#s4" target="_blank">Materials and Methods</a>. Data were segregated according to the molecular size of HA found in the lung and plotted as box and whisker plots with outliers shown as additional dots. Both NAG (A) and MPO (B) activities were significantly higher in animals that had LMW HA. Newborn pigs resuscitated with 100% O₂ and treated with NAC had significantly lower NAG (C) and MPO (D) activities than those without NAC treatment, suggesting that antioxidant treatment decreases the inflammatory response to resuscitation with hyperoxia.</p

All pigs were equally asphyxiated.

<p>All pigs were equally asphyxiated.</p

Stimulation of TNFα and IL1ß production by a 6mer HA oligosaccharide.

<p>RAW264.7 murine macrophages (1×10⁶) were stimulated with a 10 µg/ml of 6-mer HA oligosaccharide <i>in vitro</i>. Supernatant IL1ß and TNFα were measured using standard ELISA. HA6 significantly stimulated the expression of both IL1ß and TNFα in the macrophage cell line, thereby confirming that LMW HA can stimulate the expression of inflammatory cytokines in macrophages.</p

Fragmentation of HA by peroxynitrite and stimulation of TNFα and IL1ß by oligomeric HA in vitro.

<p>HMW HA (Healon™, 1×106 Da, Lane 2) was exposed to 100 µM 3-morpholinosydnonimine (SIN-1), a compound that spontaneously releases NO and superoxide to generate peroxynitrite. Exposure of HMW HA to SIN-1 results in degradation to LMW HA (300–500 kDa, Lane 6). Exposure to SIN-1 in the presence of 600 mU/ml superoxide dismutase (SOD) partially protects this degradation (Lane 7). Exposure to 300 µM PAPANOATE, a pure NO donor, does not degrade Healon™ (Lane 8). In addition, treatment of Healon™ with either 1 U/ml Streptomyces hyaluronidase at 60°C for 2 hours (Lane 4) or sonication for 2 minutes (Lane 5) results in formation of LMW HA. Just heating HMW HA at 60°C in the absence of hyaluronidase did not degrade HA in the same manner as in the presence of enzyme (Lane 2). Lane 1 has 200 kDa HA (ICN) and Lane 9 contains Hind III digested DNA makers.</p