Myron L. Seligman

Free radicals in cerebral ischemia.

The possibility that cerebral ischemia may initiate a series of pathological free radical reactions within the membrane components of the CNS was investigated in the cat. The normally occurring electron transport radicals require adequate molecular oxygen for orderly transport of electrons and protons. A decrease in tissue oxygen removes the controls over the electron transport radicals, and allows them to initiate pathologic radical reactions among cell membranes such as mitochondria. Pathologic radical reactions result in multiple products, each of which may be present in too small a concentration to permit their detection at early time periods. It is possible to follow the time course, however, by the decrease of a major antioxidant as it is consumed by the pathologic radical reactions. For this reason, ascorbic acid was measured in ischemic and control brain following middle cerebral artery occlusion. There was a progressive decrease in the amount of detectable ascorbic acid ranging from 25% at 1 hour to 65% at 24 hours after occlusion. The reduction of this normally occurring antioxidant and free radical scavenger may indicate consumption of ascorbic acid in an attempt to quench pathologic free radical reactions occurring within the components of cytomembranes.

CORTICOSTEROID (METHYLPREDNISOLONE) MODULATION OF PHOTOPEROXIDATION BY ULTRAVIOLET LIGHT IN LIPOSOMES

Abstract— UV irradiation of ovolecithin liposomes produced a dose dependent wave of peroxidation which reached a peak and then fell again coincident with substrate exhaustion. This correlated well with subsequent increases in membrane permeability. There was a progressive loss of unsaturated fatty acids, and when cholesterol was incorporated into liposomes, the UV produced a progressive loss of this steroid.

Spectrofluorescent detection of malonaldehyde as a measure of lipid free radical damage in response to ethanol potentiation of spinal cord trauma

Studies of the role of free radical damage to the spinal cord following a 400 g-cm impact have suggested an increase in at least one free radical product, malonaldehyde, 24–36 hr post injury. To investigate further the role of free radical lipid peroxidation in degeneration of the spinal cord following injury, a study of specific lipid fluorescence (SLF) indicative of the double Schiff-base adduct formed by a reaction between malonaldehyde and cellular components was carried out in the presence of ethanol, a known potentiator of free radical lipid peroxidation. The study was carried out in cats who received a 200 g-cm impact 3 hr to 23 days prior to sacrifice. Half of the impacted animals received ethanol, 5 ml/kg, prior to injury. These animals were rendered paraplegic, whereas the nonethanol treated animals were neurologically intact. Controls consisting of laminectomies alone or laminectomies with ethanol but without injury were also studied. Spinal cord segments at the impact or laminectomy site were minced and extracted with chloroform-methanol, cleared by centrifugation, and examined in a scanning fluorometer with excitation maximum at 360 nm and emission maxima at 420, 440, 450, and 460 nm. SLF was minimal in cats 3 hr and 1 day post injury, but markedly increased at 3 days. By 5 days, background levels were again found in all groups. SLF in the alcohol-pretreated impact animals rose to a peak at 7 days, followed by a decline to background by 10 days. The presence of SLF supports a role for free radical lipid peroxidation in the degenerative changes in the spinal cord following injury. The findings of two peaks of SLF activity suggest two different sites of damage. One site, found acutely after injury, appears in all groups and was associated with reversible changes, while the other site is associated with later changes and chronic paraplegia only. The two sites could be the gray and white matter.

Lipid antioxidant properties of naloxone in vitro

Summary The results of this study indicate that naloxone diminishes ironinitiated and catalyzed peroxidation in liposomes in a concentration-dependent manner. This supports the hypothesis that the action of naloxone in vivo during amelioration of spinal cord trauma and hemorrhagic shock may substantially involve its lipid antioxidant properties.

Interactions of immunoglobulins with liposomes: An ESR and diffusion study demonstrating protection by hydrocortisone

Abstract Heat-aggregated IgG, used as a surrogate for immunoglobulin configuration in immune complexes, mobilized a hydrophobic spin probe within bilayers of anionic liposomes, whereas native IgG was ineffective. Hydrocortisone, preincorporated into liposomal bilayers, both prevented the perturbation of nonpolar membrane regions and reduced the accelerated rate of solute diffusion from liposomes provoked by aggregated immunoglobulin. The capacity of aggregated, but not native, IgG to initiate membrane responses of living cells may therefore be due to its ability to alter surface bilayers or similar configurations of plasma membrane Fc receptors, events antagonized by corticosteroids.

Retarding effects of DNA on the autoxidation of liposomal suspensions

Deoxyribonucleic acid (DNA) is associated with the cell membrane of prokaryotes and the inner nuclear membrane of eukaryotes. The unsaturated fatty acids of phospholipids, which constitute the bilaminar structure of membranes, undergo autoxidation in the presence of O2. Calf thymus DNA was incubated with methyl archidonate-enriched phosphatidyl choline liposomes in order to study the effect of DNA upon the oxidation of phospholipids while present in their natural in vivo bilayer configuration. DNA retarded the rate of lipid oxidation and the TBA test, but it did not alter the induction period. These results suggest that DNA is scavenging free radicals produced within the phospholipid bilayer.

Mineralization of elastin extracted from human aortic tissues

Elastin was isolated from human aortic tissues by extraction with formic acid. The aortas came from individuals of both sexes with ages ranging from 18 to 67 years. The elastin was treated to remove pre-existing nuclei for hydroxyapatite and mineralizedin vitro by incubation in a solution metastable to hydroxyapatite. The results showed an increased rate of mineralizationin vitro as the age of the individual from whom the aorta was obtained increased. The lag period in mineral formation seen with elastin samples obtained from individuals below the age 40, disappeared with older elastin. Amino-acid analysis of the elastin samples confirmed earlier results by indicating an increase in the more polar amino acids in the older elastin samples. Exposure of the extracted elastin samples to several proteolytic enzymes including elastase confirmed the identity of the material as elastin.

Loss of essential membrane lipids and ascorbic acid from rat brain following cryogenic injury and protection by methylprednisolone.

Previous work has shown that unsaturated fatty acid components of model membrane phospholipids in vitro, damaged via a free radical mechanism, are protected by the presence of cholesterol in these membranes. The participation of these membrane lipids in the pathogenesis of traumatic injury to brain was studied in vivo using the Klatzo method of cryogenic injury in rats. Increased edema 4 hr after cryogenic injury was noted on the lesioned side. Total cerebral cholesterol was decreased significantly in the lesioned hemispheres 10 hr following injury. In lesioned animals pretreated and post-treated with methylprednisolone, there were no significant differences in the cholesterol levels. Arachidonic acid isolated from total membrane phospholipids was significantly reduced on the injured side 24 hr after injury, but not before. Other fatty acids were not significantly affected. Methylprednisolone treatment prevented the decrease in arachidonic acid. Animals that had received a cold injury had significant decreases in ascorbic acid levels after 4 hr on the lesioned side of the brain. This decrease was significantly ameliorated by corticosteroid administration. These results support the hypothesis that the protective effect of corticosteroids in cryogenic cerebral trauma may be due to antioxidant protection of major cell membrane lipid components such as cholesterol and phospholipids.

The effect of dihydroergotoxine on lipid peroxidation in vitro

Dihydroergotoxine mesylate (DHET), an ergot alkaloid derivative, is widely used to treat senile cerebral vascular insufficiency. Aspects of this age-related phenomenon may be due to deterioration by lipid oxidation of cellular membranes. DHET stabilizes EEG alpha frequencies, increases cerebral blood flow and oxygen uptake and accumulates in lipid-rich structures of the brain. The effect of DHET was studied on iron-catalyzed peroxidation of liposomes as measured by the thiobarbituric acid assay. It was found that DHET inhibits peroxidation in vitro in a dose-dependent manner. These results suggest that DHET acts in part as a lipid antioxidant when used to treat senile cerebral vascular insufficiency.