Animal Models of Tissue Ischemia to Evaluate the Importance of Oxygen in the Wound Healing Environment

Disclosure: This research was supported in part by a Dennis W. Jahnigen Career Development Scholars Award given to Dr. Gould. 

The Importance of Oxygen
Effective and dependable influx and efflux of blood, fluid, and nutrient supply is essential for wound healing. The delicate balance between adequate oxygenation and hypoxia is an important aspect of this process. Two main factors lead to hypoxia after acute injury. First, disruption of the vascular supply to the tissue decreases the amount of oxygen that can be delivered to the wound. Second, as cells become damaged or are recruited in the process of wound healing, their metabolic demand increases. The amount of oxygen that reaches the skin and participates in the wound healing process is dependent on the availability of the vascular supply to the tissue and on the oxygen tension. In order to reach the wound, oxygen must diffuse through the tissue; therefore, this process can be disrupted by an acute injury to the vascular supply, edema, or relatively low oxygen tension in the capillaries compared to the wound tissue.¹ Oxygen delivery is affected by oxygen saturation, temperature, pH, and volume status. Although hemoglobin is the major carrier of oxygen, increasing the hemoglobin concentration has little effect on oxygen delivery. Hopf et al² demonstrated no significant difference in subcutaneous oxygen tension when healthy, unanesthetized, euvolemic, euthermic volunteers had their hemoglobin level decreased to 5 g/dL compared to baseline. Unlike acute wounds, in which the subcutaneous oxygen tension usually ranges from 30 mmHg to 50 mmHg, in nonhealing chronic wounds the oxygen tension may range from 5 mmHg to 20 mmHg.³ The majority of chronic wounds that occur in the elderly human population (> 65 years) can be attributed to tissue ischemia and reperfusion injury.⁴
Maintaining normal tissue perfusion and the oxygenation that accompanies it is instrumental in wound healing and fighting infection. Prime examples of this are the richly perfused tissues of the oral cavity and the anus. Both areas are subject to repetitive trauma in the presence of high bacterial counts, yet they rarely are infected. Gottrup⁵ described 3 factors that determine the tissue partial pressure of oxygen (P_tO₂). First is the delivery of the oxygen from the lungs (oxygenation) to the tissue (circulation). While factors that affect this process are certainly important to wound healing, they are beyond the scope of this discussion. The second factor is the transport of the oxygen from the vascular system to the tissue, determined by the partial pressure of oxygen in the blood and the distance that the oxygen must travel by way of diffusion. Investigators have been interested in increasing oxygen transport as a means to accelerate wound healing since Jacques Cousteau’s divers first reported that their wounds healed faster while living 35 ft below sea level (ie, in a hyperbaric environment).⁶ Aside from the classic treatment of decompression sickness, hyperbaric oxygen (HBO) has been used for the treatment of many types of wounds, including Clostridial myonecrosis and other necrotizing infections, osteomyelitis, traumatic injuries such as crush injuries and burns, arterial insufficiency ulcers, venous stasis ulcers, diabetic foot ulcers, and chronic wounds that have failed conventional therapy.⁷ Oxygen is almost entirely bound to hemoglobin at normal atmospheric pressure. When oxygen is breathed at 2.0 to 2.4 atmospheres absolute (ATA), the excess oxygen that cannot be bound by hemoglobin remains dissolved in the plasma, resulting in partial pressures of up to 1200 mmHg.⁸ Hyperbaric oxygen has been shown to enhance neovascularization and fibroblast proliferation.^9,10 Bonomo et al¹¹ and Zhao et al¹² have postulated that oxygen delivered via HBO may act as a signal transducer rather than a simple nutrient because of a synergistic effect when ischemic wounds were treated with PDGF and HBO. Ironically, hypoxia, while detrimental to wound healing, also appears to be an important signal to initiate the wound healing process. For example, the transcription factor hypoxia-inducible factor-1 (HIF-1), which is not normally active, is upregulated during periods of decreased oxygen tension, stimulating the upregulation of genes that participate in angiogenesis and glucose utilization.^5,13
The third factor that determines the P_tO₂ is the consumption of oxygen in the tissue.¹⁴ Oxygen dependent enzymes, or oxygenases, play a critical role in oxygen utilization during wound healing. Oxygenases depend on oxygen and PtO2 for the enzymatic incorporation of oxygen into substrates that are involved in many of the body’s metabolic pathways. Increasing the partial pressure of oxygen (PO_²) above the maximum saturation of hemoglobin leads to an increased P_tO₂, and thus a greater availability of oxygen that can be utilized by these enzymes. Oxygenases are involved in multiple wound healing activities. Some regulate the rate of collagen deposition, while others repress the activity of hypoxia-inducible factor (HIF)-1a or augment angiogenesis. In order to form a stable collagen triple helix, proline and lysine in the procollagen molecule undergo hydroxylation by the oxygenases prolyl and lysyl hydroxylase.¹⁵ Hypoxia prevents stable collagen deposition because prolyl hydroxylase requires an oxygen tension of at least 20 mmHg to perform at 50% of its capacity.^16,17 Another oxygenase that is critical for wound healing and for the immune defense system is the nicotinamide adenine dinucleotide phosphate (NADPH)-linked oxygenase of leukocytes, also known as NADPH oxidase. This enzyme is responsible for the respiratory burst when leukocytes engulf microorganisms. When NADPH oxidase is activated it increases the oxygen consumption of leukocytes, monopolizing much of the oxygen that is found in the wound environment, causing production of oxidants and lactate.⁶ Increased lactate levels can also result from anaerobic glycolysis in leukocytes and macrophages that populate the wound environment. Allen et al¹⁸ showed that for NADPH oxidase to work at 50% of its maximum speed, the oxygen tension must be between 40 mmHg and 80 mmHg. The bactericidal effect of leukocytes is due to the production of superoxides, which is highly oxygen dependent. Therefore, decreased oxygen tension reduces the efficacy of the leukocytes, resulting in higher bacterial counts in contaminated wounds, with increased potential for bacterial invasion.^19,20
The metabolism of oxygen results in the production of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion, hydroxyl radical, and hypochlorite ion. Both hypoxia and hyperoxia are implicated in the production of ROS, which appear to be key regulators of many wound healing activities.²¹ For example, both keratinocytes and macrophages increase vascular endothelial growth factor (VEGF) production when treated with H₂O₂.^22,23 H₂O₂ can also act as a signaling molecule for the activation of signal transducers and activators of transcription (STATs) that target genes, which are involved in a myriad of processes such as cell growth, apoptosis, and antioxidant defense.²⁴

The Quest for the Perfect Ischemic Wound Model
Although valuable for examining mechanisms at a molecular level, in-vitro cell culture models cannot replicate the complex environment of a wound. Factors that affect the wound healing process and are difficult to simulate in vitro include: variations in temperature, pH, circulation, O₂ tension, and nutrient supply/utilization. While each of these can be adjusted individually, the culture environment tends to have a greater supply of nutrients and a less stressful chemical environment than patients will ever experience.²⁵ Perhaps the most important feature that is lacking in cell culture is the interaction of multiple cell types in 3 dimensions. Therefore, to truly understand the process of wound healing and the variations that occur in wound pathology, one must rely on animal models. Animal models have been utilized to study radiation and angiogenesis,²⁶ antiseptics and the immunologic response,²⁷ microcirculation,²⁸ and to examine the effect of oxygen.²⁹ However, there is no model of a truly chronic wound that mimics wounds occurring in humans. Recently, several models to mimic pressure ulcers have been described.^30–32 Their utility as chronic wound models remains to be explored. Meanwhile, a lack of animal models for venous insufficiency and diabetic ulcers remains.³³ Thus, the quest for a stable, reliable, and reproducible animal wound model that mimics the chronic wound state continues. Many investigators have devoted significant effort to develop ischemic wounds in animal models because ischemia and ischemia-reperfusion appear to be common denominators among all chronic wounds. The findings are then extrapolated to similar wounds in humans.
Hunt-Schilling chamber. One of the earliest methods utilized to examine the biochemical changes in the wound environment was the Hunt-Schilling chamber. Schilling et al³⁴ first described his removable stainless steel wire mesh chamber in 1959 and it was subsequently utilized by Hunt et al³⁵ in their studies of respiratory gas and pH in healing wounds. Because the device is subcutaneous, extraction of cells, wound fluid and connective tissue can be performed without disruption of the chamber. Growth factors can be injected directly into the chamber and samples can be removed over time to examine the in-vivo response. Likewise, the chamber can be employed to observe the effect of oxygen on the wound healing response. Lactate levels measured in the chamber fluid can be compared to systemic levels as a means to determine the amount of ischemia present in the wound.³⁶ Technically simple, the subcutaneously implanted wound chambers are placed via blunt dissection, usually on the dorsum of rodents, with minimal surgical training required. Although it provides for temporal sampling of the “wound” over an extended period, the Hunt-Schilling chamber does not produce an open, chronic, ischemic wound that resembles human pathology. With respect to the study of oxygen, the wound-healing environment surrounding an implanted device may not truly replicate the chronic wound environment in vivo. To realistically mimic the chronic wounds of humans, the authors believe it is important to reduce both the oxygen tension and the local blood flow to the wound.

McFarlane flap. The McFarlane flap is probably the most well known and frequently modified animal model used to study the effect of compromised circulation. Usually designed as a dorsal skin flap on rodents, the original flap was 4 cm in width and 10 cm in length, located in between the scapular tips and the hip joints, designed to provide a length-to-width ratio that was approximately 2.5 to 1 (Figure 1.³⁷The flap can be modified to be cranially or caudally based in an H-shaped fashion.³⁸ The original McFarlane flap did not have open wounds within the flap itself. McFarlane et al³⁹ initially evaluated these flaps for the presence or absence of necrosis and later examined the reorientation of the circulation within the flap and the delay phenomenon. In their examination of the cranially-based McFarlane flap, Hurn et al⁴⁰ noted that regardless of whether the flap was delayed or not, almost all of the flaps became necrotic distally. In addition, the authors also noted that central necrosis is sometimes seen in this flap. The central necrosis was explained by the hunched stance that rats often assume after undergoing the procedure. This kyphotic positioning initiates maximum stress on the central portion of the flap, which when combined with the pressure of dressing results in compromise to the circulation in the central region with subsequent necrosis, and may account for the distal necrosis. Utilizing flaps created on the ventral surface of rats, Hallock⁴¹ determined that flap viability was dependent upon the origin and quality of the blood supply and not whether it was cranially or caudally based. The McFarlane flap and its modifications are technically simple and easily reproducible with minimal training of ancillary staff. The entire flap can be utilized for tissue samples or sectioned to study the effects of hypoxia, hyperoxia, and the biochemical factors involved in the wound healing process within the borders of the flap. Areas lateral to the flap can be used as internal, nonischemic controls as long as they are in a similar craniocaudal location.
Bipedicle flap. In an effort to examine the effects of an ischemic gradient in the wound healing process, Schwarz et al⁴² developed a bipedicle flap based on the concept of the McFarlane flap.³⁹ Schwarz et al used a 10-cm x 4-cm flap that was elevated on the dorsum of the rats to the level of the panniculus carnosus and then immediately secured with surgical staples. Excisional wounds were created 4 days after the flaps were elevated. This model revealed signs of delayed wound healing without tissue necrosis, in that control wounds healed by 8 days after injury, while wounds in the experimental group were not healed until day 12. Histologic analysis revealed decreased thickness of both dermis and epidermis with a concomitant lack of new collagen synthesis. The design of this model allows for the measurement of subcutaneous oxygen tension in areas not involved in the flap while simultaneously measuring oxygen tension within the flap. Similar to the McFarlane flap, the biochemical and histologic effects of hypoxia and hyperoxia on the wound-healing environment can be examined through obtaining tissue samples from the flap. To increase the ischemic insult and delay the wound healing further, Chen et al⁴³ modified the Schwarz bipedicle flap model by extending the length to 11 cm and decreasing the width to 2.5 cm. The modified Schwarz bipedicled flap model is easily reproducible with minimal training of ancillary staff. Excisional wounds can be evaluated as well as the flap itself. The entire flap can be utilized for tissue samples or sectioned to study the effects of hypoxia within the borders of the flap.
Although these flaps have been used since the 1960s, very few investigators have validated whether the flaps are truly ischemic or determined how long they remain ischemic. Using implanted electrodes in the dermis, Gould et al⁴⁶ demonstrated that while the Chen modification of the McFarlane flap is no longer ischemic after 2 weeks, excisional wounds placed in the center of the flap heal more slowly than controls. In addition to vascular flow from the cranial and caudal pedicles, inosculation, and neovascularization can come from the wound bed, supplying additional nutrients and blood supply. This can significantly alter tissue perfusion and duration of ischemia. Mechanisms to block the influence of the underlying wound bed include suturing the flap along its length to form a closed tube and closing the wound bed, or inserting a barrier, such as a silicone sheet or wafer, between the flap and the wound bed.^44,45 Gould et al⁴⁶ documented biochemical and mechanical evidence of tissue ischemia by decreasing the width of the flap to 2.0 cm and placing an intervening sheet of silicone between the flap and the dorsum of the rat. Although flow does not necessarily equate with tissue oxygenation, the perfusion in the flap can be measured by laser Doppler flow or labeled microspheres. Studies designed to evaluate vascular flow through elevated pedicled flaps have shown that although flow through the pedicle is preserved, flow to the tip of the flap may be reduced to less than 20% of initial values within the first 12 hours. This gradually returns to within 75% of normal by 1 to 2 weeks after flap elevation, and to 100% by 3 to 4 weeks post elevation.^47–50 Vascular flow can be altered by vasoconstriction or vasodilation due to temperature and compression from edema or dressings. This may account for some of the inter-animal and inter-investigator variability seen in these animal models.
Another important variable that distinguishes the rodent model from humans is the degree of wound contraction. Because rodents are loose-skinned animals, their wounds heal primarily by contraction. Efforts to combat this include splinting the wound with silicone or other devices placed under or on top of the wound. How to correlate this to the amount of contraction that occurs in humans needs further investigation.

The ischemic rabbit ear model. The ischemic rabbit ear ulcer model is perhaps one of the most extensively validated methods for examining the importance of oxygen in the healing wound (Figure 2). The blood flow characteristics and the vascular pattern in the rabbit ear can easily be examined with backlighting. Ahn and Mustoe⁵¹ described an ischemic rabbit ear model in which the effects of oxygen and tissue ischemia can be evaluated in the setting of an ischemic ulcer—wounds in this model healed within 2 weeks due to the development of collateral circulation. However, with re-ligation of the vessels, this wound model may simulate chronically ischemic wounds. The ischemic rabbit ear model has been used to determine how growth factors affect the wound-healing environment. In the model developed by Ahn and Mustoe,⁵¹ TGF-b1 mRNA was shown to be increased for up to 12 days after the initial hypoxic injury when compared to control groups. This model has been used to explore the effect of hypoxia and hyperoxia on TGF-b1, PDGF, VEGF, and bFGF.⁵² It has also been applied to examine the effect of aging on ischemic wound healing.^53,54 In addition to following the fate of endogenous growth factors, the model has been used to study the effect of topical application of exogenous recombinant growth factors. These studies suggest that the difference between ischemic wound healing in young and old rabbits may be due to an altered response to growth factors, particularly TGF-b.⁵⁴
Standardized excisional full-thickness dermal wounds are created via punch biopsy in each ear down to the auricular cartilage. To make a wound that has an avascular base, care must be taken to remove the perichondrium without disrupting the underlying cartilage. If the cartilage is damaged, granulation tissue can form that actually originates from the skin on the opposite side of the cartilage.³³ One distinguishing characteristic between this model and the other models described is that the rabbit ear does not heal by wound contraction. Thus, the rabbit ear model more closely resembles the granulation type healing process that occurs in humans. The ligation of 2 of the 3 arteries supplying the ear, as described by Ahn and Mustoe,⁵¹ is technically demanding and requires surgical skill and magnification. However, this modification allows the investigator to mimic more closely ischemic wounds that occur in the human population. The ischemic rabbit ear model provides a large surface area in which multiple wounds and tissue samples, such as granulation tissue, can be obtained from a single animal for the study of oxygen and its effects on the wound-healing environment. Rabbits are exquisitely sensitive to anesthesia, are significantly more expensive than rats and mice, and require more resources for their housing, dietary needs, and handling. Although conceptually a similar model could be applied to rats and mice, the technical aspects have made this prohibitive.
Ischemic limb. Partial or total ligation of the major vessels to a limb can be performed in animal models to induce ischemia that simulates peripheral arterial occlusive disease. Critical limb ischemia can be induced in one hind limb, while the contralateral hind limb can be utilized as a nonischemic control. In the rat, the common iliac artery, femoral artery, and their associated branches are usually completely ligated to produce the effects of limb ischemia. Utilizing this technique, Paek et al⁵⁵ described a hind limb model that was demonstrably ischemic, including reduced muscle PO₂ for up to 40 days. This model has been used to study the effect of exogenous vascular endothelial growth factor (VEGF). Also using the rat model, Chang et al⁵⁶ found that oxygen tension in experimentally induced ischemic limbs was normalized to that of nonischemic controls at 4 and 8 weeks after VEGF administration. Ischemic limb models are technically demanding and are complicated by inter-animal variation. Collateral vessel formation, which normalizes the blood flow to the ischemic limb, may occur at varying rates leading to differences in experimental measurements. Obtaining experimental data and tissue is also more complex than in previously described animal models of wound ischemia. Even noninvasive means of measuring vascular flow are variable, such as Doppler measurements. Obtaining tissue at various time points and at various distances distal to the point of ischemia can also vary from animal to animal. Protecting the ischemic limb from cannibalism makes following the ischemic wounds over time problematic.

Rationale for the Ischemic Bipedicle Flap Model

The authors chose to focus on the rodent model because the animals are small, easy to handle, cost effective, and easily attainable. Using a modification of the Schwarz model, the flap was progressively narrowed and a silicone barrier added to prevent vessel ingrowth from the rat’s dorsum (Figures 3 and 4). The final product is a flap that does not develop necrosis, yet, remains ischemic for up to 2 weeks with markedly impaired wound healing. Although quite flexible, by suturing to both the skin flap and to the dorsal muscle fascia, the silicone sheet also acts as a splint, preventing wound contraction to some extent.
The model was standardized to reduce inter-animal variation and to be technically straightforward, so that trained personnel with minimal surgical experience could perform the model with good reproducibility and flap survival. The rats tolerate the flap well and can be treated with systemic or topical agents. The wounds can be measured and excised, providing sufficient tissue for multiple biochemical analyses. The model was initially performed and validated in Sprague-Dawley rats. The flap was subsequently utilized as a model of age and ischemia. It was found that Fisher 344 rats are more susceptible to ischemia, necessitating a further modification of the flap dimensions (unpublished data, 2007). In addition, the same principle was applied to mice, with the caveat that wound excision was more difficult, requiring loupe magnification, and necrosis with silicone exposure was more frequent (unpublished data, 2007). The Sprague-Dawley ischemic flap model has been used to examine whether hyperbaric oxygen improves wound healing through ROS-mediated signaling.⁵⁷ It was demonstrated that HBO improves the rate of wound closure in ischemic wounds but has no effect on wound closure rate in nonischemic wounds (Figure 5).⁵⁷ Using biochemical and histologic analyses, it was demonstrated that apoptosis is attenuated by HBO treatment of ischemic wounds and that this effect is blocked by systemic administration of the free radical scavenger, N-acetylcysteine (NAC). Furthermore, reduced inducible nitric oxide synthase (iNOS), reduced 3-nitrotyrosine, and induction of copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase, and glutathione peroxidase in the hyperbaric treated ischemic wounds, indicating modulation of the balance between ROS and endogenous antioxidants.⁵⁸ Although it is not a chronic wound model, the authors believe that this model has the potential to be a valuable tool for understanding the contribution of ischemia to impaired wound healing.