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Wound Healing and Hyperbaric Oxygen Therapy Physiology: Oxidative Damage and Antioxidant Imbalance

One of the basic pathways to non-healing of wounds is the interplay between tissue hypo perfusion, resulting hypoxia, and infection. Chronic hypoxia both within the wound and periwound environment impedes wound healing by numerous mechanisms that act in a concurrent fashion. One of the challenges of advanced wound care is identifying the extent to which local hypoxia contributes to the abnormal healing, and then correcting that hypoxia to the extent possible. During the past 40 years, a large body of research and clinical evidence has been accumulated demonstrating that intermittent oxygenation of hypo perfused tissue, which can only be achieved by exposure to hyperbaric oxygen (HBO), mitigates many of these impediments and sets in motion a cascade of responses that contributes to wound healing.1

Wound Care

Adequate molecular oxygen is required for a wide range of biosynthetic processes essential to normal healing. Molecular oxygen is required for hydroxylation of proline during collagen synthesis and cross linking as well as provisional of substrate for the production of reactive oxygen species during the respiratory burst occurring within leukocytes that phagocytizes bacteria. While short-term hypoxia is one stimulus for angiogenesis in wound healing, adequate local oxygen levels are required to sustain an effective angiogenic response and for the reconstruction of the dermal matrix. Recent research has shown that oxygen also plays an important role in cell signaling events necessary for tissue repair, which further explains the fragile dynamic between oxygen availability and increased demands for oxygen during wound healing.2

Hyperbaric Oxygen Environment:

Hyperbaric oxygenation of tissue is achieved when a patient breathes 100% oxygen in an environment of elevated atmospheric pressure typically in the ranging of 2.0 to 3.0 ATA (atmospheres absolute). This can occur in a monoplace (single patient) chamber typically compressed with 100% oxygen or less frequently in a multiplace (multiple patient) chamber typically compressed with air with the patient breathing 100% oxygen through a specially designed hood or mask. Both can increase PO2 values in excess of 1700 mmHg.3, 4

Oxygen is transported by the blood in two different ways: (1) chemically bound to hemoglobin in erythrocytes and (2) physically dissolved in plasma according to Henry’s law, the gas tension of oxygen in blood and tissue will increase as the partial pressure of oxygen increases in the alveoli. This will lead to increased oxygen availability through increased oxygen transport dissolved in the plasma and ultimately in the tissues.

Wound Care

Hemoglobin transport of oxygen is limited by chemical binding were as the hemoglobin concentration of oxygen dissolved in plasma is only limited by the partial pressure of oxygen in the alveoli which is determined by the partial pressure of oxygen in the inspired gas. At three ATA the amount of oxygen dissolved in plasma can theoretically reach ≈ 2240 mmHg, an amount large enough to maintain life in the absence of hemoglobin. This was demonstrated experimentally in 1960 by Boerema who, by exchange transfusion with Ringer’s lactate, exsanguinated the blood from young pigs while inside a hyperbaric chamber. In the absence of hemoglobin, the pigs survived on only the oxygen physically dissolved in their plasma.5

When the body is at rest, it normally consumes about 6 ml of oxygen per 100 ml of blood, but of this amount only 0.3 ml is transported by hemoglobin in the red blood cells. When the pressure is raised to 2 ATA of pure oxygen, the plasma oxygen level is raised to 4.4 ml. Thus, oxygen saturation of the tissue is considerably enhanced with the use of hyperbaric oxygen therapy. At 3 ATA approximately 6.4 volumes percent of oxygen are physically dissolved in plasma, which is sufficient to sustain life even in the absence of hemoglobin.

When the amount of dissolved oxygen in plasma is increased so does the diffusion distance of oxygen from the capillaries. The oxygen diffusion distance increases approximately 4 fold on the arterial end (64-247 micrometers) of the capillary and doubles that distance at the venous end (36-64 micrometers) with the increased PaO2 from breathing air at 1 ATA and 100% oxygen at 3 ATA.6

Hyperbaric oxygen therapy (HBOT) provides additional oxygen to the hypoxic tissue and supports tissue healing through a variety of mechanisms. The immediate effects of HBOT occurring during treatments improve wound metabolism in the setting of acute and chronic hypoxia. These relatively short lived effects in support of wound healing include the following: (1) Improved local tissue oxygenation, leading to improved cellular energy metabolism. (2) Increased collagen and other extracellular matrix protein deposition and epithelization. (3) Decreased local tissue edema due to vasoconstriction of vessels in nonischemic tissues. (4) Improved leukocyte bacterial killing and suppressed exotoxin production. (5) Increased effectiveness of antibiotics that require oxygen for active transport across microbial cell membranes. These effects, while important, would not by themselves account for the degree of improvement in wound healing seen in most ulcers treated with HBOT.

Over the past 15 years, research has led to a somewhat different understanding of the role of HBOT, which has focused on the role of HBOT plays in altering the balance of reactive oxygen (ROS) and reactive nitrogen (RNS) species within the wound, fundamentally altering the wound environment and its healing response. In this context, HBOT also must be thought of as providing oxygen as a cell-signaling agent.7,8 Achieving these beneficial effects requires a minimum tissue oxygen tension of approximately 200 mmHg which can only be achieved with use of HBO therapy, these effects include: (1) Enhanced growth factor and growth factor receptor site production, especially platelet derived growth factor (PDGF) and vascular endothelial growth factor. (VEGF), which are helpful in wound matrix development and angiogenesis. (2) Altered leukocyte β-integrin receptor sensitivity and is helpful in mitigating ischemia reperfusion injuries, which occurs in many chronic and acute wounds. (3) Reduces inflammation and apoptosis which occurs in many acute ischemic wound models. (4) Activated stem cell metabolism and their release into circulation from bone marrow reservoirs. These physio-pharmacological changes have been observed both in vitro and in vivo.9-12

In effect, HBOT is complicated by the dual nature of oxygen in being essential to life, and at the same time, it’s toxicity in excess. Hyperoxygenation enhances ROS and RNS mediated pathways, which can be expressed as both positive and negative. While the body’s defenses against excessive ROS is highly effective, it can be overcome with prolonged exposures at high oxygen tensions, as occurs with HBOT. This leads to one of the greatest impediment to the application of HBOT which is neurologic and pulmonary manifestations of oxygen toxicity. Oxygen tolerance limits that avoid these manifestations are well defined. To negate these negative effects the application of HBOT is limited to 3 ATA of 100% oxygen. Acute and chronic wounds are generally treated at 2.0 to 2.5 ATA at these levels daily exposures do not produce pulmonary symptoms.3

Reactive oxygen species are a normal by-product of cellular metabolism whose rate of production is increased in the presence of increased oxygen availability, as occurs during an HBO exposure.13 Not only primary, but also secondary generation of reactive oxygen intermediated molecules add to oxidant injury such as in neutrophil oxidant production.

The role of HBOT in free radical mediated tissue injury is not well defined. Researchers have demonstrated that HBOT enhances antioxidative defence mechanisms in some animal studies. HBO has also been reported to increase the production of ROS. Hyperoxia causes an increase in nitric oxide (NO) synthesis as part of a response to oxidative stress. Mechanisms for neuronal nitric oxide synthase (nNOS) activation include augmentation in association with a chaperone protein called heat shock protein 90 (Hsp90) and intracellular entry of calcium.14

Superoxide Dismutase (SOD) is an important enzyme found in human cells that inactivates superoxides, the most common free radicals in the body, responsible for destruction of cells. HBO stimulates SOD production thereby helping the body to rid itself of the byproducts of inflammation and damaging free radicals.

Wound Environment:

Acute wound healing is an orderly and efficient process which is characterized by four distinct, but overlapping phases: hemostasis, inflammation, proliferation and remodeling. In non-healing chronic wounds this efficient and orderly process has been lost due to the absence of the hemostasis phase, causing these ulcers to become locked into a state of chronic inflammation that is self-sustaining and characterized by abundant neutrophil infiltration with associated reactive oxygen species and destructive enzymes. Healing can only occur after this prolonged inflammatory phase is broken and the wound’s micro-wound environment is once again in balance, thus allowing the wound to proceed through the remaining stages15,16 (Fig 1).

All chronic wounds are similar in that each is characterized by one or more persistent inflammatory stimuli: repetitive trauma, ischemia, or low-grade bacterial contamination. Once the skin barrier is broken and bacterial colonization occurs, inflammatory molecules from bacteria such as endotoxin, platelet products such as transforming growth factor β (TGF-β) or fragments of extracellular matrix (ECM) molecules such as fibronectin stimulate proliferation of inflammatory cells (neutrophils and microphages) to enter the wound. These activated inflammatory cells secrete proinflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β).16

These proinflammatory cytokines (TNFα and IL-1β) also synthesize matrix metalloproteinase (MMP) and suppress tissue inhibitors of matrix metalloproteinase (TIMP).16 The chronic wound environment has been proven to contain elevated protease (MMP) levels and decreased levels of protease inhibitors (TIMP).16 Matrix metalloproteinases play an important role in all phases of wound healing by promoting cell migration, breaking down extracellular matrix, and remodeling. An imbalance in the microwound environment with increasing numbers of MMP’s and decrease TIMP’s levels is associated with the degradation of collagen and growth factors and growth factor receptor sites as well as other vital components of the extracellular matrix. Once growth factors are degraded, communication between the various cells participating in the wound healing process stops and wound healing is delayed. As the inflammatory cycle is prolonged it amplifies the pro-inflammatory cytokine cascade leading to wound fluid which has been found to be absent of DNA synthesis. This is related to the low mitotic activity, excessive levels of inflammatory cytokines, high levels of proteases, and excessive reactive oxygen species found in chronic wound fluid which in turn results in senescent or mitotically incompetent cells.17-19

Once the inflammatory cycle is prolonged, it creates a “vicious cycle” which is characteristic of all chronic wounds.16 The self-sustaining nature of this vicious cycle has far reaching effects. As the wound shifts from hypoxic to ischemic over time the lack of available oxygen for metabolism leads to anaerobic metabolism which reduces concentrations of adenosine triphosphate (ATP) resulting in metabolic acidosis. The reduction of ATP facilitates increased lactate with decreasing pH which excites nociceptors and produces activation of pH-sensitive ion channels, resulting in pain. Likewise, if there is sufficient oxygen for aerobic metabolism, then the by-product acid is metabolized into carbon monoxide and water, which is the final step of complete metabolism. As a result, pain is alleviated or disappears. This accounts for why acute wounds have lower levels of associated pain when compared to chronic wounds.20-22

Free radicals are known to cause cell damage and function as inhibitory factors in the healing process.20 The production of reactive oxygen species (ROS) associated within a chronic wound can originate from several potential sources. During healing various inflammatory cells; neutrophils, macrophages, endothelial cells, fibroblasts, and in particular senescent fibroblasts which are prominent in chronic wounds, are all capable of produce superoxide.15 But, in chronic wounds activated neutrophils and macrophages produce extremely large amounts of superoxide and its derivatives via the phagocytic isoform of NADPH oxidases.21 When polymorphonuclear neutrophils (PMNs) are recruited and activated at the wound site they consume an increased amount of oxygen, which is converted into ROS, in a process known as a “respiratory” or “oxidative” burst. This burst requires the consumption of large amounts of molecular oxygen, increasing oxygen consumption by at least 50%.22 Resulting in the generation of superoxide anions. Most of the superoxide anions formed are converted into hydrogen peroxide.17 Some of the hydrogen peroxide (H2O2) is converted into highly toxic hydroxyl radicals via the iron-catalysed Fenton reaction, creating a second source of ROS. Iron is released from hemoglobin by degraded erythrocytes, ferritin, and hemosiderin.24 An example of this is venous ulcerations which have excessive iron deposition in the skin, which is used in a Fenton reaction to produce excessive amounts of ROS, in this case hydroxyl radicals. These highly toxic hydroxyl free-radicals also enhance the synthesis and activation of even more matrix-degrading metalloproteinase.25 The presence of excessive reactive oxygen metabolites are not only highly toxic to surrounding tissue, they also increase MMPs while decreasing TIMP levels, creating a highly aggressive chronic wound environment that inhibits healing.


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