Expression of T helper cell–associated inflammatory mediator mRNAs in cells of bronchoalveolar lavage fluid samples and oxygen concentration in arterial blood samples from healthy horses exposed to hyperbaric oxygen

Maty G. P. Looijen Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.

Search for other papers by Maty G. P. Looijen in
Current site
Google Scholar
PubMed
Close
 DVM
,
Dallas J. New Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.

Search for other papers by Dallas J. New in
Current site
Google Scholar
PubMed
Close
 DVM, MSc
,
Carrie D. Fischer Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.

Search for other papers by Carrie D. Fischer in
Current site
Google Scholar
PubMed
Close
 PhD
,
Rkia Dardari Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.

Search for other papers by Rkia Dardari in
Current site
Google Scholar
PubMed
Close
 PhD
,
Karyn M. Irwin Moore Equine Veterinary Services Ltd, 260048A Writing Creek Crescent, Rocky View County, AB, T4A 0M9, Canada.

Search for other papers by Karyn M. Irwin in
Current site
Google Scholar
PubMed
Close
 DVM
,
Christopher J. Berezowski Moore Equine Veterinary Services Ltd, 260048A Writing Creek Crescent, Rocky View County, AB, T4A 0M9, Canada.

Search for other papers by Christopher J. Berezowski in
Current site
Google Scholar
PubMed
Close
 DVM
,
Stephanie L. Bond Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.

Search for other papers by Stephanie L. Bond in
Current site
Google Scholar
PubMed
Close
 BVSc, BA Anim Sci
, and
Renaud Léguillette Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.
Moore Equine Veterinary Services Ltd, 260048A Writing Creek Crescent, Rocky View County, AB, T4A 0M9, Canada.

Search for other papers by Renaud Léguillette in
Current site
Google Scholar
PubMed
Close
 DVM, PhD

Abstract

OBJECTIVE To evaluate the mRNA expression of T helper (Th)1, Th2, and Th17 cell–associated inflammatory mediators in cells of bronchoalveolar lavage fluid samples collected from healthy horses exposed to hyperbaric oxygen (HBO) and to monitor blood oxygen concentration during and following HBO therapy.

ANIMALS 8 healthy horses.

PROCEDURES In a randomized controlled crossover design study, each horse was exposed (beginning day 1) to 100% oxygen at a maximum of 3 atmospheres absolute (304 kPa) daily for 10 days or ambient air at atmospheric pressure in the HBO chamber for an equivalent amount of time (control). Bronchoalveolar lavage fluid samples were collected on days 0 and 10. After validation of candidate reference genes, relative mRNA expressions of various innate inflammatory, Th1 cell–derived, Th2 cell–derived (including eotaxin-2), Th17 cell–derived, and regulatory cytokines were measured by quantitative PCR assays. For 3 horses, arterial blood samples were collected for blood gas analysis during a separate HBO session.

RESULTS The optimal combination of reference genes was glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine ribosyltransferase, and ribosomal protein L32. Compared with day 0 findings, expression of eotaxin-2 mRNA was significantly lower (0.12-fold reduction) and the percentage of neutrophils in bronchoalveolar lavage fluid samples was significantly lower on day 10 when horses received HBO therapy. Values of Pao2 rapidly increased (> 800 mm Hg) but immediately decreased to pretreatment values when HBO sessions ended.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HBO therapy does not increase mRNA expression of inflammatory cytokines, but reduces eotaxin-2 mRNA transcription. The Pao2 increase was transient with no cumulative effects of HBO.

Abstract

OBJECTIVE To evaluate the mRNA expression of T helper (Th)1, Th2, and Th17 cell–associated inflammatory mediators in cells of bronchoalveolar lavage fluid samples collected from healthy horses exposed to hyperbaric oxygen (HBO) and to monitor blood oxygen concentration during and following HBO therapy.

ANIMALS 8 healthy horses.

PROCEDURES In a randomized controlled crossover design study, each horse was exposed (beginning day 1) to 100% oxygen at a maximum of 3 atmospheres absolute (304 kPa) daily for 10 days or ambient air at atmospheric pressure in the HBO chamber for an equivalent amount of time (control). Bronchoalveolar lavage fluid samples were collected on days 0 and 10. After validation of candidate reference genes, relative mRNA expressions of various innate inflammatory, Th1 cell–derived, Th2 cell–derived (including eotaxin-2), Th17 cell–derived, and regulatory cytokines were measured by quantitative PCR assays. For 3 horses, arterial blood samples were collected for blood gas analysis during a separate HBO session.

RESULTS The optimal combination of reference genes was glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine ribosyltransferase, and ribosomal protein L32. Compared with day 0 findings, expression of eotaxin-2 mRNA was significantly lower (0.12-fold reduction) and the percentage of neutrophils in bronchoalveolar lavage fluid samples was significantly lower on day 10 when horses received HBO therapy. Values of Pao2 rapidly increased (> 800 mm Hg) but immediately decreased to pretreatment values when HBO sessions ended.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HBO therapy does not increase mRNA expression of inflammatory cytokines, but reduces eotaxin-2 mRNA transcription. The Pao2 increase was transient with no cumulative effects of HBO.

Contributor Notes

Address correspondence to Dr. Léguillette (rleguill@ucalgary.ca).
  • 1. Gill AL, Bell CN. Hyperbaric oxygen: its uses, mechanisms of action and outcomes. QJM 2004;97: 385395.

  • 2. Slovis N. Review of equine hyperbaric medicine. J Equine Vet Sci 2008;28: 760767.

  • 3. Tibbles PM, Edelsberg JS. Hyperbaric-oxygen therapy. N Engl J Med 1996;334: 16421648.

  • 4. Dennog C, Radermacher P, Barnett YA, et al. Antioxidant status in humans after exposure to hyperbaric oxygen. Mutat Res 1999;428: 8389.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Thom SR. Oxidative stress is fundamental to hyperbaric oxygen therapy. J Appl Physiol 2009;106: 988995.

  • 6. Buras JA, Reenstra WR. Endothelial-neutrophil interactions during ischemia and reperfusion injury: basic mechanisms of hyperbaric oxygen. Neurol Res 2007;29: 127131.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Benson RM, Minter LM, Osborne BA, et al. Hyperbaric oxygen inhibits stimulus-induced proinflammatory cytokine synthesis by human blood-derived monocyte-macrophages. Clin Exp Immunol 2003;134: 5762.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Inamoto Y, Okuno F, Saito K, et al. Effect of hyperbaric oxygenation on macrophage function in mice. Biochem Biophys Res Commun 1991;179: 886891.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Weisz G, Lavy A, Adir Y, et al. Modification of in vivo and in vitro TNF-α, IL-1, and IL-6 secretion by circulating monocytes during hyperbaric oxygen treatment in patients with perianal Crohn's disease. J Clin Immunol 1997;17: 154159.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Lahat N, Bitterman H, Yaniv N, et al. Exposure to hyperbaric oxygen induces tumour necrosis factor-alpha (TNF-α) secretion from rat macrophages. Clin Exp Immunol 1995;102: 655659.

    • Search Google Scholar
    • Export Citation
  • 11. Cimsit M, Uzun G, Yıldız S. Hyperbaric oxygen therapy as an anti-infective agent. Expert Rev Anti Infect Ther 2009;7: 10151026.

  • 12. Narkowicz CK, Vial JH, McCartney PW. Hyperbaric oxygen therapy increases free radical levels in the blood of humans. Free Radic Res Commun 1993;19: 7180.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Clark JM, Lambertsen CJ. Rate of development of pulmonary O2 toxicity in man during O2 breathing at 2.0 Ata. J Appl Physiol 1971;30: 739752.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Oter S, Korkmaz A, Topal T, et al. Correlation between hyperbaric oxygen exposure pressures and oxidative parameters in rat lung, brain, and erythrocytes. Clin Biochem 2005;38: 706711.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Demchenko IT, Welty-Wolf KE, Allen BW, et al. Similar but not the same: normobaric and hyperbaric pulmonary oxygen toxicity, the role of nitric oxide. Am J Physiol Lung Cell Mol Physiol 2007; 293: L229L238.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Jackson RM. Pulmonary oxygen toxicity. Chest 1985;88: 900905.

  • 17. Jamieson D, Chance B, Cadenas E, et al. The relation of free radical production to hyperoxia. Annu Rev Physiol 1986;48: 703719.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18. Jamieson DD. Lipid peroxidation in brain and lungs from mice exposed to hyperoxia. Biochem Pharmacol 1991;41: 749756.

  • 19. Baumwart CA, Doherty TJ, Schumacher J, et al. Effects of hyperbaric oxygen treatment on horses with experimentally induced endotoxemia. Am J Vet Res 2011;72: 12661275.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Dhar M, Neilsen N, Beatty K, et al. Equine peripheral blood-derived mesenchymal stem cells:isolation, identification, trilineage differentiation and effect of hyperbaric oxygen treatment. Equine Vet J 2012;44: 600605.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Holder TE, Schumacher J, Donnell RL, et al. Effects of hyperbaric oxygen on full-thickness meshed sheet skin grafts applied to fresh and granulating wounds in horses. Am J Vet Res 2008;69: 144147.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Shaw FL, Handy RD, Bryson P, et al. A single exposure to hyperbaric oxygen does not cause oxidative stress in isolated platelets: no effect on superoxide dismutase, catalase, or cellular ATP. Clin Biochem 2005;38: 722726.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. O'Connor AM, Sargeant JM, Gardner IA, et al. The REFLECT statement: methods and processes of creating reporting guidelines for randomized controlled trials for livestock and food safety by modifying the CONSORT statement. Zoonoses Public Health 2010;57: 95104.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Couëtil LL, Rosenthal FS, DeNicola DB, et al. Clinical signs, evaluation of bronchoalveolar lavage fluid, and assessment of pulmonary function in horses with inflammatory respiratory disease. Am J Vet Res 2001;62: 538546.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. Fogarty U, Buckley T. Bronchoalveolar lavage findings in horses with exercise intolerance. Equine Vet J 1991;23: 434437.

  • 26. Hoffman AM. Bronchoalveolar lavage: sampling technique and guidelines for cytologic preparation and interpretation. Vet Clin North Am Equine Pract 2008;24: 423435.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Wasko AJ, Barkema HW, Nicol J, et al. Evaluation of a risk-screening questionnaire to detect equine lung inflammation: results of a large field study. Equine Vet J 2011;43: 145152.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28. Fernandez NJ, Hecker KG, Gilroy CV, et al. Reliability of 400-cell and 5-field leukocyte differential counts for equine bronchoalveolar lavage fluid. Vet Clin Pathol 2013;42: 9298.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. Beekman L, Tohver T, Dardari R, et al. Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease. BMC Mol Biol 2011;12: 5.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Beekman L, Tohver T, Leguillette R. Comparison of cytokine mRNA expression in the bronchoalveolar lavage fluid of horses with inflammatory airway disease and bronchoalveolar lavage mastocytosis or neutrophilia using REST software analysis. J Vet Intern Med 2012;26: 153161.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. Giguère S, Prescott JF. Quantitation of equine cytokine mRNA expression by reverse transcription-competitive polymerase chain reaction. Vet Immunol Immunopathol 1999;67: 115.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32. Ruijter JM, Ramakers C, Hoogaars WM, et al. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 2009; 37: e45.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33. Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002; 3:RESEARCH0034.

    • Search Google Scholar
    • Export Citation
  • 34. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 2002; 30: e36.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35. Freeman BA, Crapo JD. Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria. J Biol Chem 1981;256: 1098610992.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36. Plafki C, Peters P, Almeling M, et al. Complications and side effects of hyperbaric oxygen therapy. Aviat Space Environ Med 2000;71: 119124.

    • Search Google Scholar
    • Export Citation
  • 37. Corrigan C. The eotaxins in asthma and allergic inflammation: implications for therapy. Curr Opin Investig Drugs 2000;1: 321328.

  • 38. White JR, Imburgia C, Dul E, et al. Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils. J Leukoc Biol 1997;62: 667675.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39. Weston MC, Collins ME, Cunningham FM. Equine CCL11 induces eosinophil cytoskeletal reorganization and activation. Inflamm Res 2006;55: 4652.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40. Benarafa C, Collins M, Hamblin A, et al. Role of the chemokine eotaxin in the pathogenesis of equine sweet itch. Vet Rec 2002;151: 691693.

    • Search Google Scholar
    • Export Citation
  • 41. Weston MC, Cunningham FM, Collins ME. Distribution of CCR3 mRNA expression in horse tissues. Vet Immunol Immunopathol 2006;114: 238246.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42. Ravensberg AJ, Ricciardolo FL, van Schadewijk A, et al. Eotaxin-2 and eotaxin-3 expression is associated with persistent eosinophilic bronchial inflammation in patients with asthma after allergen challenge. J Allergy Clin Immunol 2005;115: 779785.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43. Jenkinson SG, Jordan JM, Duncan CA. Effects of selenium deficiency on glutathione-induced protection from hyperbaric hyperoxia in rat. Am J Physiol 1989; 257: L393L398.

    • Search Google Scholar
    • Export Citation
  • 44. Niu K-C, Huang W-T, Lin M-T, et al. Hyperbaric oxygen causes both antiinflammation and antipyresis in rabbits. Eur J Pharmacol 2009;606: 240245.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45. Pablos MI, Reiter RJ, Chuang J-I, et al. Acutely administered melatonin reduces oxidative damage in lung and brain induced by hyperbaric oxygen. J Appl Physiol 1997;83: 354358.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46. Barber RD, Harmer DW, Coleman RA, et al. GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues. Physiol Genomics 2005;21: 389395.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 47. Bogaert L, Van Poucke M, De Baere C, et al. Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids. BMC Biotechnol 2006;6: 24.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48. Cappelli K, Felicetti M, Capomaccio S, et al. Exercise induced stress in horses: selection of the most stable reference genes for quantitative RT-PCR normalization. BMC Mol Biol 2008;9: 49.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 49. Figueiredo MD, Salter CE, Andrietti AL, et al. Validation of a reliable set of primer pairs for measuring gene expression by real-time quantitative RT-PCR in equine leukocytes. Vet Immunol Immunopathol 2009;131: 6572.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 50. Pfaffl MW. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 2001; 29: e45.

  • 51. Benedetti S, Lamorgese A, Piersantelli M, et al. Oxidative stress and antioxidant status in patients undergoing prolonged exposure to hyperbaric oxygen. Clin Biochem 2004;37: 312317.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 52. Freeman BA, Topolosky MK, Crapo JD. Hyperoxia increases oxygen radical production in rat lung homogenates. Arch Biochem Biophys 1982;216: 477484.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 53. Perng W-C, Wu C-P, Chu S-J, et al. Effect of hyperbaric oxygen on endotoxin-induced lung injury in rats. Shock 2004;21: 370375.

  • 54. Deaton CM, Marlin DJ, Smith NC, et al. Effect of acute airway inflammation on the pulmonary antioxidant status. Exp Lung Res 2005;31: 653670.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 55. Kirschvink N, Smith N, Fievez L, et al. Effect of chronic airway inflammation and exercise on pulmonary and systemic antioxidant status of healthy and heaves-affected horses. Equine Vet J 2002;34: 563571.

    • Search Google Scholar
    • Export Citation

Advertisement