PURPOSE: The lack of suitable experimental models of chronic severe limb ischemia and deficiencies in the available methods that allow for direct intermittent measurement of regional limb perfusion are obstacles to the evaluation of recently developed molecular strategies to reverse severe limb ischemia. Our aim was to develop a model of clinically relevant severe limb ischemia and correlate a simple direct measurement of muscle pO(2) to a clinical ischemia index, muscle mass, and capillary density. METHODS: Severe hindlimb ischemia was induced in 44 adult rats with ligation of the left common iliac artery, the femoral artery, and their branches. The effect of ischemia on muscle pO(2) was measured in the left gastrocnemius with room air and with 100% oxygen at 3, 10, 24, and 40 days after ischemia was induced. Clinical ischemia index, muscle mass, cellular proliferation, and capillary density also were assessed. RESULTS: The clinical ischemia index of the left limb was most severe at day 10, with evidence of pressure sores, a pale and dusky limb, and abnormal gait. With the rats breathing room air, muscle pO(2) was significantly lower in the left limbs than in the right limbs at days 3, 10, 24, and 40. After an oxygen challenge (100% O(2)), muscle pO(2) was significantly lower at 3, 10, and 40 days. At 3 days, the fraction of muscle mass per total body weight of the left tibialis anterior (TA) was significantly greater than the right TA as a result of edema and inflammation. By days 10, 24, and 40, the left gastrocnemius and TA masses were significantly less than the right as a result of muscle atrophy. Histopathology showed severe necrosis in the left gastrocnemius and TA on day 3. Inflammation was greatest by day 10. Necrotic muscle regenerated but remained atrophic at 40 days. The TA was slower to recover than the gastrocnemius. Capillary densities and capillary-to-muscle fiber ratios were greater in the ischemic limb than in the normal limb at day 24. Cellular proliferation as determined with bromodeoxyuridine labeling reagent staining was maximal in the ischemic limb at day 3. CONCLUSION: We have developed a rat model of chronic severe hindlimb ischemia with persistent ischemia as shown with a simple direct measurement of muscle pO(2) for up to 40 days. This model of severe hindlimb ischemia may be applicable for future studies of molecular strategies to treat severe limb ischemia in humans.

Animals↗

PubMedDate not supplied

Hyperoxia improves microvascular perfusion in a murine wound model.

There is a need for a noninvasive method that measures wound angiogenesis. Hyperoxia is known to increase the appearance of new blood vessels in wounds, yet no study has confirmed increases in wound bed perfusion with periodic hyperbaric oxygen (HBO) exposure. This study investigates whether laser Doppler imaging is able to detect and quantify the enhancement of wound angiogenesis that is known to occur with intermittent HBO treatments. Full-thickness dorsal dermal wounds were created on mice randomized to hyperoxic (n = 14) and control (n = 15) groups. Hyperbaric oxygen was administered twice daily for 90 minutes each at 2.1 atmospheres for 7 days. Wound bed perfusion was measured by laser Doppler imaging on days 0, 7, and 10 postwounding. Wound blood flow increased significantly over baseline on day 7 and 10 in the hyperoxic group, but only on day 10 in the control group. Comparison between groups showed a 20% statistically significant increase in wound perfusion in HBO-treated animals compared to control on day 10 (p = 0.05). Laser Doppler imaging was able to detect and quantify the increase in wound bed perfusion resulting from intermittent HBO treatments and shows promise as a noninvasive measure of angiogenesis and wound healing.

Animals↗

PubMedDate not supplied

Hyperoxia and angiogenesis.

We hypothesized that tissue hyperoxia would enhance and hypoxia inhibit neovascularization in a wound model. Therefore, we used female Swiss-Webster mice to examine the influence of differential oxygen treatment on angiogenesis. One milliliter plugs of Matrigel, a mixture of matrix proteins that supports but does not itself elicit angiogenesis, were injected subcutaneously into the mice. Matrigel was used without additive or with added vascular endothelial growth factor (VEGF) or anti-VEGF antibody. Animals were maintained in hypoxic, normoxic, or one of four hyperoxic environments: hypoxia -- 13 percent oxygen at 1 atmosphere absolute (ATA); normoxia -- 21 percent oxygen at 1 ATA; hyperoxia -- (groups a-d) 100 percent oxygen for 90 minutes twice daily at the following pressures: Group a, 1 ATA; Group b, 2 ATA; Group c, 2.5 ATA; Group d, 3.0 ATA. Subcutaneous oxygen tension was measured in all groups. The Matrigel was removed 7 days after implantation. Sections were graded microscopically for the extent of neovascularization. Angiogenesis was significantly greater in all hyperoxic groups and significantly less in the hypoxic group compared with room air-exposed controls. Anti-VEGF antibody abrogated the angiogenic effect of both VEGF and increased oxygen tension. We conclude that angiogenesis is proportional to ambient pO(2) over a wide range. This confirms the clinical impression that angiogenesis requires oxygen. Intermittent oxygen exposure can satisfy the need for oxygen in ischemic tissue.

Animals↗