Effect of Oxygen Concentration on the Formation of Malondialdehyde-Like Material in a Model of Tissue Ischemia and Reoxygenation

This study was conducted to explore the functional relationship between oxygen concentration during tissue reoxygenation after ischemia and the extent of postischemic lipid peroxidation, an indicator of reoxygenation injury. Excised rat liver or kidney tissue was rendered ischemic for 1 h at 37°C, minced into 1 mm3 fragments, and then reoxygenated for 1 h in flasks of buffered salt solution containing various amounts of oxygen. Production of malondialdehyde-like material (MDA) was measured to indicate lipid peroxidation. MDA production was minimal at oxygen tensions less than 10 mmHg, increased sharply from 10 to 50 mmHg, and plateaued at approximately 100 mmHg. A similar functional relationship was produced by a simple mathematical model of free radical mediated lipid peroxidation in biological membranes, suggesting that MDA production is indeed caused by free radical oxidation of membrane phospholipids and that the oxygen effect is governed by simple competition between chain propagation and chain termination reactions within the membrane. These experimental and analytical results confirm that relatively low concentrations of oxygen arc sufficient to produce oxidative damage in post-ischemic tissues

A Rapid, Widely Applicable Screen for Drugs that Suppress Free Radical Formation in Ischemia/Reperfusion

Substantial injury can occur during reoxygenation of previously ischemic tissue in many experimental models, as the result of the generation of oxygen-derived free radicals. To test the antiradical activity of potentially protective compounds in this setting, we developed a simple screening system, applicable to fresh biopsy specimens, in which warm ischemia and reoxygenation of excised tissue are performed in vitro. Tissue production of malondialdehyde (MDA) equivalents is used as a nonspecific-but-sensitive marker of oxygen radical damage. Test compounds with putative antiradical activity are added prior to the reoxygenation phase, and their ability to suppress MDA production is an index of activity in preventing reoxygenation injury. Comparison with ischemic but not reoxygenated controls confirms the oxygen-dependent nature of the effect. Standard positive controls of known effective agents, such as butylated hydroxytoluene or deferoxamine, provide a reference for the activity of the test compound. The method is applicable to surgical biopsy specimens in veterinary and human medicine

Simulating Low-Frequency Sonic Pulsations to Achieve Thrombolysis

Cardiovascular thrombosis may result in critical ischemia to a range of anatomical regions, constituting a leading cause of death in the United States. Current invasive treatments for such arterial blockages often yield blood clot recurrence, resulting in repeated hospitalization of patients. This research aims to show how internally introduced pressure oscillations may be used to initiate thrombolysis. We present a novel computational model for determining the resonant frequency and corresponding deformation of an idealized thrombus. Sinusoidal pressure differences across the thrombus induce axial displacements of frequency dependent amplitude. The maximum peak displacement occurs at a resonant frequency of 73 Hz for 2 mm radius clot and 140 Hz for a smaller, scaled 1.3 mm radius clot. For a larger, scaled 2.5 mm radius clot, a resonant frequency of 67 Hz induced maximum displacement. Strains exceeding 160%, a value sufficient for clot lysis, occurred at only ±1.0 mmHg axial pressure gradients at 73 Hz (2 mm radius). This simple test case constitutes preliminary feasibility for the concept of vibration induced thrombolysis. Finally, we are left with a convincing indication that internal ultrasonic pulsation may be employed for degrading proximal and distal clot fragments

Simple Methods for Determining the Accuracy of Tumor Blood Flow Measurements Using Radioactive Microspheres in Rats

Two simple methods are presented that allow positive identification of the accuracy and precision of the microsphere technique and a quick verification of sphere entrapment in tumor vessels. A known flow of Ringer\u27s solution from a motor-driven syringe is perfused through the rat\u27s isolated systemic circulation from left ventricle to right atrium and collected in a funnel. Using this preparation, total blood flow in rats measured with radioactive microspheres injected into the left ventricle was 97% of actual flow. The coefficient of variation (standard deviation/mean) of the microsphere measurements was 0.22. In the same preparation, non-entrapment of microspheres in subcutaneous tumor nodules grown on a hind limb could be measured from the difference in counts collected in venous effluent before and after placement of a tourniquet proximal to the tumor. For example, in two types of transplantable carcinoma, we found non-entrapment of less than 0.1% of the injected microspheres. Such a shunt would correspond to less than 10% of microspheres entering a typical tumor nodule and, in turn, less than 10% underestimation of true flow to the tumor. These two techniques may be helpful to other investigators in testing the accuracy of microsphere methods in various small animal tumor models

Methylene Blue as an Inhibitor of Superoxide Generation by Xanthene Oxidase: A Potential New Drug for the Attenuation of Ischemia/Reperfusion Injury

Tissue oxidases, especially xanthine oxidase, have been proposed as primary sources of toxic oxygen radicals in many experimental models of disease states. Among these, ischemia-reperfusion injury may be of the greatest clinical interest. In this paper we propose the use of methylene blue as a means of suppressing the production of superoxide radicals, O2, by acting as an alternative electron acceptor for xanthine oxidase. Previous work has indicated that methylene blue accepts electrons from xanthine oxidase at the iron-sulfur center. Initial experiments in our laboratory demonstrated that (1) pairs of electrons from each enzymatic oxidation are transferred to methylene blue, (2) the reduction of methylene blue can be achieved by model iron-sulfur centers, similar to the iron-sulfur center of xanthine oxidase, (3) reduced methylene blue auto-oxidizes to produce H2O2 directly, rather than O2, and (4) methylene blue is effective at non-toxic levels (2-5 mg/kg) in preventing free radical damage to liver and kidney tissues in an in vitro model of ischemia and reoxygenation. Accordingly, we propose that methylene blue may represent a new class of antioxidant drugs that competitively inhibit reduction of molecular oxygen to superoxide by acting as alternative electron acceptors for tissue oxidases. We have termed these agents “parasitic” electron acceptors

Quantitative Effects of Iron Chelators on Hydroxyl Radical Production by the Superoxide-Driven Fenton Raction

Iron bound to certain chelators is known to promote the conversion of superoxide radicals (O2) to hydroxyl radicals (HO\u27) by the superoxide-driven Fenton reaction. The production of HO\u27 by various iron chelates was studied using the reaction of dimethyl sulfoxide and HO\u27 to produce methane sulphinic acid. Methane sulphinic acid was quantified by use of a simple colorimetric assay and used to determine the amounts of HO\u27 produced. Superoxide was generated from 200 M hypoxanthine and 0.05 U/ml xanthine oxidase in the presence of 0-100 M iron and 100 M of each chelator. The results of this preliminary investigation illustrate that, at physiological pH, the superoxide-driven Fenton reaction is significantly promoted by iron chelated to EDTA, nitrilotriacetate, and citrate, but is not promoted by the other anions studied

Evidence That Free Fatty Acid-Iron Complexes Directly Initiate Lipid Peroxidation in Vitro and in Vivo: A New Mechanism of Oxidative Stress

Through a series of biochemical and histochemical experiments we explored the novel hypothesis that iron and free fatty acids, liberated after tissue injury, combine to form liposoluble complexes that directly initiate lipid peroxidation. The addition of 100 M ferric iron to 30 mM linoleate suspensions at pH 7.4 produced time dependent lipid peroxidation, measured as conjugated diene formation. Complexes of 100 M ferric iron and 600 M pentanoate also initiated formation of conjugated dienes in linoleate suspensions and formation of malondialdehyde-like materials in rat liver slices. A histochemical stain for free fatty acids revealed positive reactions within cell membranes in traumatized regions of rat liver tissue that underwent compression injury followed by thirty minutes of blood perfusion, but not in nontraumatized control regions. The diaminobenzidine-H2O2 histochemical reaction for iron, revealed increased levels of redox cyclable iron in the membranes and the cytoplasm of traumatized hepatocytes. We propose that traumatic injury initiates cascades leading to liberation of iron from storage proteins and free fatty acids from membranes, which combine, distribute to the lipid domains of cell membranes, and directly initiate lipid peroxidation

Abnormal Response of Tumor Vasculature to Vasoactive Drugs

The effects of the vasoconstrictor, phenylephrine, and the vasodilator, hydralazine, on blood flow to tumor were studied and compared to those on blood flow to normal tissues in vivo. Regional blood flow and cardiac output were measured with the use of radioactive microspheres in 150- to 250 g inbred Harlan F344 rats bearing subcutaneous nodules of two types of transplantable carcinoma ( hard and soft ) with microscopically different vascular patterns. Three groups of rats were treated with hydralazine, saline, or phenylephrine, and regional blood flow was determined at the time of maximum blood pressure response. Results were correlated with quantitative morphometric analysis of arteriolar and capillary wall thickness in tumor and normal tissue. Phenylephrine decreased, and hydralazine increased, normal tissue perfusion as indicated by cardiac output. Tumor blood flow remained low and was not significantly influenced by drug treatment, except for the phenylephrine effect on hard tumors. Histological study of tumor vessel walls revealed· an absence of smooth muscle capable of responding to the vasoactive drugs by constriction or dilation. Evidently, by their selective action on normal vessels, vasoactive drugs can change the ratio of tumor to normal tissue perfusion. In particular, the increase of normal tissue vs. tumor blood flow by vasodilator drugs may enhance the selectivity of local heat therapy

Characterization of the Oscillometric Method for Measuring Indirect Blood Pressure

In this study, human subjects and dogs were used to determine the ability of the oscillometric method to indicate systolic and diastolic pressure. In the human studies, the auscultatory method was used as the reference. In the animal studies, directly recorded blood pressure was used as the reference. The ability of the sudden increase in cuff pressure oscillations during cuff deflation to indicate systolic pressure was examined and found to overestimate systolic pressure slightly in man, but more in animals. Systolic pressure was encountered when the cuff pressure oscillations were about one half of their maximum amplitude. However, in both man and animals the ratio was not constant; although the range was less in man than in animals. Diastolic pressure was encountered when cuff pressure oscillation amplitude was about 0.8 of the maximal amplitude. This ratio for diastolic pressure was not constant over a range of diastolic pressure. The range of variability was less for man than for the dog