Physicians Corner

Oxygen under pressure: for improved healing

Oxygen: The New Growth Factor?

Oxygen under pressure: for improved healing
Hyperbaric oxygen therapy can provide pharmacological doses of oxygen to healing tissue. In a typical HBOT treatment, hyperbaric oxygen is capable of providing tissue oxygen levels of greater than 11 times normal, or up to 620 mmHg. Most chronic wounds are hypoxic and HBOT provides the oxygen needed to stimulate and support healing. Some examples of typical levels of oxygenation provided in a hyperbaric oxygen treatment are illustrated in Table 1:

When used in wound healing HBOT provides a short pulse of oxygen - 90 minutes in a 24-hour day. Although, such a short time provision of elevated oxygen could not significantly effect wound healing, HBOT acts in numerous ways that affect the wound after the treatment has stopped. There are seven principal methods in which HBOT is capable of affecting tissue:

1. Vasoconstrictive effects of oxygen
2. 100% oxygen concentration effects on the diffusion gradient
3. Hyperoxygenation of ischemic tissue
4. Down regulation of inflammatory cytokines
5. Up-regulation of growth factors
6. Leukocyte effects
7. Antibacterial effects

Vasoconstrictive effects of oxygen: The vasoconstrictive effects of HBO2 can be used to good effect to treat patients. HBOT causes a significant reduction of edema, which has been shown to be beneficial in reperfusion injury, crush injury, compartment syndrome, burns, wound healing, and failing flaps.

Oxygen diffusion effects: The diffusion of nitrogen out of the tissues in decompression sickness is facilitated by the use of 100% oxygen. In wound healing the beneficial effects of oxygen are primarily related to the concentration of oxygen molecules in the tissue, rather than by diffusion kinetics. However, the rate of oxygen entry into the wound environment is affected by the rate of diffusion from the capillaries. Edema adversely affects the achievement of high oxygen concentrations in the wound and increases the inter-capillary diffusion distance. Even a small increase in tissue edema can dramatically slow the rate of entry of oxygen into the tissues and can cause tissue hypoxia.

Hyperoxygenation of tissue: The oxygenation of hypoxic tissue is one of the key mechanisms by which HBOT accelerates wound healing. Numerous studies have shown a dose response curve for the provision of oxygen in the wound healing environment. However, oxygen is a powerful drug and just like other drugs, it is possible to give too little or too much. Chronic wounds are frequently hypoxic and the provision of HBOT corrects the hypoxia, albeit temporarily. It then allows for acceleration of the wound healing process through processes which continue long after the HBOT session has ended and tissue oxygen levels have returned to pre-treatment values. Over time the oxygenation of the chronic wound improves with HBOT therapy. Marx and Johnson demonstrated that for irradiated wounds, HBO2 induces neovascularization, which becomes significant after about 14 treatments and continues for years after the HBOT therapy has ceased. A typical chronic wound will usually require 20 to 30 HBOT treatments. This probably represents the amount of neovascularization needed to sustain wound healing.

Cytokine down regulation and growth factor up-regulation: HBO is capable of favorably influencing a number of cytokines and growth factors important to wound healing. When administered after wounding, HBO up-regulates collagen synthesis through pro-al(I) mRNA expression. In rabbit ear wounds HBOT has been shown to up-regulate mRNA for the platelet-derived growth factor (PDGF)-beta receptor. In ischemic flaps HBO up-regulates fibroblast growth factor (FGF) causing an increased effect over that seen with FGF alone. In situations where FGF is ineffective, HBO can render it highly effective, although the effect is different from up-regulation. In patients with Crohn's disease interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF)-alpha levels are diminished during HBO treatment. TNF levels in normal rats become elevated after a single exposure to HBO. For different physiological conditions HBO may cause up- or down regulation of cytokines. Vascular endothelial growth factor (VEGF) is up-regulated by hypoxia, yet the hyperoxia of HBO also up-regulates this factor. The effects of transforming growth factor (TGF)-beta1 and platelet-derived growth factor (PDGF)-beta are synergistically enhanced by HBO

Oxygen and infection

Oxygen is key to the phagocytosis and killing of bacteria by neutrophils or polymorphonuclear cells (PMNs). This process involves the production of oxygen radicals and superoxides and is directly influenced by the oxygen concentration in the tissue. As the oxygen tension falls below 30 mmHg the efficiency of bactericidal action of PMNs begins to drop off dramatically. This was demonstrated by Knighton et al in 1984 where the phagocytic activity of neutrophils in ingesting Staph. aureus was compared to oxygen tension. The activity level of phagocytosis is shown in.

PMN-mediated killing of several aerobic bacteria - Proteus vulgaris, Salmonella typhimurium, Klebsiella pneumoniae, Serratia marcescens, Pseudomonas aeruginosa and Staphylococcus species - is diminished in hypoxia. Increasing the concentration of oxygen over ambient levels has been shown to reduce infection. When supplemental oxygen was administered during a surgical operation and for two hours postoperatively, infection rates dropped by as much as 54%. Thus, increasing tissue concentrations of oxygen has a beneficial effect on the ability of PMNs to combat bacteria and prevent infection.

HBO and infection

HBO2 has six actions which have been used to combat clinical infection:

1. Tissue rendered hypoxic by infection is supported
2. Neutrophils are activated and rendered more efficient
3. Macrophage activity is enhanced
4. Bacterial growth is inhibited
5. Release of certain bacterial endotoxins is inhibited
6. The effect of antibiotics is potentiated.

   Support of infected hypoxic tissue: Soft tissue and bone infections are frequently accompanied by localized areas of tissue hypoxia caused by the inflammatory processes accompanying infection and by subsequent vascular thrombosis. As the infected tissue becomes infiltrated with inflammatory cells (PMNs and platelets) the PO2 falls. In clostridial gas gangrene the production of phospholipase C has been associated with platelet and neutrophil aggregation and vascular thrombosis with subsequent hypoxia. Administration of HBO can cause the PO2 to increase five-fold in infected tissue.

   Neutrophil activation: As tissue PO2 rises, leukocyte killing of bacteria becomes much more efficient. Below a PO2 of 30 mmHg PMN killing is markedly reduced. Because areas of hypoxia accompany serious tissue infections, HBO is an effective means of raising tissue PO2 to levels at which PMNs can function effectively. By raising tissue PO2 to levels higher than that achieved by breathing oxygen at ambient pressure, bacterial killing by PMNs is further enhanced. Thus, by increasing tissue oxygen tension, a better than 'normal' antibacterial effect can be achieved. Hunt and colleagues have demonstrated that the clearance of bacteria from hypoxic tissue is enhanced by hyperoxic breathing mixtures

   .

   Figure 6 - Wound bacterial growth and oxygen tension

   Enhancement of macrophage activity: Macrophages, like PMNs, are affected by tissue oxygen tension. They perform a key role in combating infection by scavenging bacteria and foreign material. Under hypoxic conditions macrophages are unable to scavenge effectively and produce peroxides . Hypoxia also induces macrophages to produce the inflammatory cytokines TNF-alpha, IL-1, IL-8, and intracellular adhesion molecule-1, which can adversely affect the response to infection . While it is not yet known whether HBO can enhance macrophage function, it may be needed to bring the PO2 of hypoxic tissue up to normal levels so that macrophages can function normally.

   Inhibition of bacterial growth: Anaerobic bacteria are particularly susceptible to increased concentrations of oxygen. The more sensitive the anaerobic organism is to oxygen, the lower the level of superoxide dismutase, an enzyme that allows cells to defend themselves against oxygen free radicals. With HBO large amounts of oxygen free radicals can be generated, making anaerobic bacteria particularly susceptible to oxidative killing.

   Inhibition of endotoxin release: In C. perfringens infections the major source of tissue injury and death is caused by the alpha toxin. Secretion of this toxin is suppressed by HBO. In rats exposed to E. coli peritonitis, HBO administration increased survival from 0% to 92%. One of the mechanisms by which HBO appears to have worked is by antagonizing some of the harmful effects of bacterial endotoxin release. However, to be optimally effective, HBOT must be given early in the course of infection and combined with appropriate surgical debridement and antibiotics.

   Potentiation of antibiotics: Both Knighton et al and Hunt et al have demonstrated that oxygen adds to the effectiveness of antibiotics; the greater the concentration of oxygen, the more pronounced the effect. This has been demonstrated in experimental Staph. aureus osteomyelitis treated with cefazolin. In Pseudomonas aeruginosa infections HBO2 has an additive effect with aminoglycoside antibiotics, reducing morbidity and mortality.

   

   Figure 7 - Potentiation of antibiotic effects by HBO

   Conclusion

   Hyperbaric oxygen is a powerful treatment for acute and chronic wounds, acting on injured and healing tissue in a number of ways. Hypoxic tissue, reperfusion injury, compartment syndrome, crush injury, failing flaps, chronic wounds, burns and necrotising infections have all been shown to respond favorably to HBO. As we learn more about how HBO benefits wounds by up-regulating growth factors, down regulating cytokines, reducing edema, and supporting angiogenesis and new tissue growth, the potential benefits to wound healing become clearer.

   References

   Available on request

    

   Oxygen: The New Growth Factor?

   In recent years, our understanding of the intercellular communication of healing has increased considerably. Cells within a wound receive a myriad of signals from their environment, the sum of which governs the activity of a cell. The term "cytokine" is applied to those substances, which function as cellular signals. Growth factors are a subclass of cytokines that specifically stimulate the proliferation of cells. This stimulation may occur through several different mechanisms. For example, some growth factors have activities that attract fibroblasts and inflammatory cells, some act as mitogens, stimulating cell division, and some effect the production and degradation of the extra-cellular matrix. All of these phenomenon are the result of a cytokine (growth factor) signaling the cell nucleus to produce proteins, which account for the observed activities. A clear understanding of growth factor physiology carries the promise of clinical advances in wound management. Currently one cytokine, Platelet Derived Growth Factor, is in clinical use for the management of problem wounds. As our knowledge of these substances expands, other growth factors will be added to our clinical armamentarium for the management of non-healing wounds.

   Non-healing wounds can also be managed by optimizing the metabolic requirements of healing e.g. protein, trace elements, and oxygen. The most frequent common denominator in non-healing wounds is inadequate tissue oxygenation, which impairs healing and host defenses. Correction of such hypoxia by means of revascularization or hyperbaric oxygen therapy results in healing for most patients. Conventional wisdom suggests that oxygen is just a metabolite and therefore healing, in these circumstances, is simply a reflection of having sufficient oxygen to meet the energy demands of wound repair. However, some exciting evidence is now emerging to suggest that oxygen serves a dual role as both a metabolite and a growth factor. The conceptualization of oxygen as a growth factor has considerable relevance to the field of hyperbaric oxygen therapy.

   The idea of oxygen acting as a cell signal has already been established in the setting of hypoxia. As an example, gene expression for erythro-protein production is largely proportional to the pO2 level in the kidney. It has been proposed that cells in a non-healing wound may respond to hyperbaric therapy because the supra-physiologic elevation of tissue oxygen serves as a trigger signaling that enough oxygen is in the environment to proceed with normal healing. Subsequent daily exposure to the threshold oxygen level reinforces this signal and results in gene expression of the protein building blocks required for healing. It makes sense for cells to conserve resources until the environmental signals are strong enough and consistent enough to activate the cell nucleus and begin the healing process.

   Two separate groups of investigators have published findings that support this concept of oxygen as a growth factor. Following a single one-hour exposure to hyperbaric oxygen, Hehenberger, et al. (1997) demonstrated a dose dependent stimulation of normal in vitro fibroblasts with a peak increase in cell proliferation at 2.5 ATA O2. The dose-dependent effect of a single 1-hour exposure to oxygen suggests a pharmacologic effect of oxygen on cells, as opposed to an increased metabolic availability of oxygen. These findings suggest, therefore, that a single brief exposure to hyperbaric oxygen on a daily basis provides a strong initiating signal for the intracellular events that culminate in cell proliferation, while sustained hyperoxia has the opposite effect.

   In a study of in vitro fibroblast proliferation using tritium labeled thymidine, Tompach, et al., found that a single dose of HBO (2.4 ATA for 120 minutes) produced a sustained stimulation of fibroblasts for 72 hours. If a second exposure to HBO was given on the same day there was no additional increase in cell proliferation. Similarly, cultured endothelial cells remained stimulated for 72 hours following a single 15-minute exposure to HBO. Again, these findings suggest that we must reconsider oxygen as being more than just a metabolite.

   This new paradigm of oxygen as a growth factor is consistent with the clinical observation that a BID dosing of HBO appears to offer no clear benefit over a QD dosing schedule for the treatment of chronic wounds. As our understanding of oxygen physiology increases, we will be in a better position to determine the optimal dosing of oxygen in both its metabolic and stimulatory roles.