Abstract

Hyperbaric oxygen (HBO) therapy is the intermittent inhalation of 100% oxygen at a pressure greater than one atmosphere absolute. It is an effective treatment for various inflammatory conditions, including chronic wounds which are characterized by an excessive influx of neutrophils and their prolonged persistence at the wound site. Neutrophil apoptosis and clearance have been shown to be required for resolution of inflammation. The mechanisms by which HBO aids wound healing are well documented, but its effects on cellular inflammatory response are not well understood particularly with respect to neutrophils. The hypothesis presented in this thesis is that increased oxygenation via HBO assists chronic wound healing by enhancing non-inflammatory neutrophil defences and cell death through apoptosis. An investigation was carried out into the effects of HBO on neutrophil antimicrobial function and apoptosis using differentiated HL-60 cells as an in vitro neutrophil model. The data clearly showed that a single HBO treatment for 90 min caused an increase in the oxidative burst activity of neutrophil-like cells as shown by increased NBT staining, superoxide (cytochrome c reduction) and H2O2 production (Kruskal-Wallis, P < 0.05), and phagocytosis of Staphylococcus aureus. HBO treatment displayed a pro-apoptotic effect, enhancing caspase 3/7 activity both in the presence and absence of a TNF-α stimulus (Kruskal-Wallis, P < 0.05) and causing morphological changes (observed using Giemsa and SYBR® Safe staining) associated with apoptosis. Although no consistent pattern was observed, both hyperoxia and pressure alone seemed to contribute to both the increase in antimicrobial activity and the increase in apoptosis induced by HBO in these neutrophil-like cells (Chapters 4 and 5). HBO-enhanced neutrophil clearance by macrophages was investigated using bovine neutrophils and monocyte-derived macrophages (MDMФ). A single 90 min HBO exposure significantly increased the clearance of fresh and 22 h-aged neutrophils by MDMФ (two-way ANOVA, P < 0.05), suggesting an increase in phosphatidylserine (PS) exposure in apoptotic neutrophils after HBO treatment (Chapter 6). Importantly, a long-term repetitive exposure to HBO in patients with chronic wounds caused a significant decrease in the antioxidant enzyme defence system (one-way repeated measures ANOVA, P < 0.05), plasma TNF-α and IL-1β after 30 HBO sessions, with down regulation of expression of the anti-apoptotic factors, NF-B and Bcl-2 (Chapter 7). These findings may go some way towards explaining the effectiveness of HBO treatment not only for chronic wounds but also for other inflammatory conditions that may be affected by this treatment.

Keywords

Chronic wound, Hyperbaric oxygen therapy, Neutrophils, Inflammation

Document Type

Thesis

Publication Date

2013

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