| dc.description.abstract | Hyperbaric oxygen therapy (HBOT) could induce oxidative stress over time that may cause lung inflammation and alter the mRNA expression of inflammatory cytokines in lung cells of horses.
Identifying stable reference genes is necessary to obtain reliable relative quantitative polymerase chain reaction (QPCR). We hypothesized that (1) HBOT induces lung inflammation in healthy horses and (2) hyperbaric oxygen (HBO) induces an increase in arterial oxygen levels with no other effect on arterial blood parameters.
Eight horses were used in a randomized controlled cross-over design. Treated horses were exposed to 100% oxygen at 3 atmosphere absolute (ATA) for 20 minutes for 10 days whereas the chamber was not pressurized for control horses. A bronchoalveolar lavage (BAL) was performed at baseline and on day 10 for total and differential cell counts as well as for the mRNA expression. Groups of pre- and post-HBOT and control were compared. IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IFN-γ, TNF-α and Eotaxin-2 were measured by QPCR in BAL fluid. Genes’ expression was measured by QPCR after efficiency correction using relative expression software tool (REST) software analysis. The expression stability of four candidate reference genes, GAPDH, HPRT, SDHA, RPL-32, were determined using NormFinder and Genormplus. Arterial blood parameters were measured before and right after HBOT on day 1 and 10. Four additional horses were used to measure arterial blood gases collected through an arterial line during HBOT at baseline, 3 ATA (for 0, 10 and 20 minutes), during (2 ATA) and 0 and 10 minutes after decompression.
Results show that HBOT induced a significant decrease in total and neutrophilic cell counts for the HBOT pre vs. post groups. The mRNA expression of cytokines was significantly down- regulated with HBOT for Eotaxin-2 (HBO post vs. control post) and IL-4 (HBO pre vs. post). GAPDH was found to be the most stable reference gene. The number of reference genes used for optimal normalisation included GAPDH and HPRT or RPL-32. Arterial blood parameters during HBOT showed a rapid increase of PaO2 (>800mmHg), which decreased to baseline values within 10 minutes after HBO.
These results suggest that HBO reaches extremely high blood oxygenation levels very transiently and does not induce inflammation in the lungs of horses. | |
