Harry B. Demopoulos

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.

Experimental spinal cord injury: treatment with naloxone.

We studied the effect of the opiate antagonist naloxone on the recovery of cats injured with a 400-g-cm impact injury to T-9. The animals were evaluated by recording somatosensory evoked potentials and performing weekly neurological examinations. Several dose schedules were followed. Six of eight cats that received an intravenous or intraperitoneal bolus of naloxone (10 mg/kg) 45 minutes after injury regained the ability to walk. Recovery occurred in only one of five animals that were treated with an infusion of naloxone, 10 mg/kg/hour, and in none of five animals given 1 mg/kg as a bolus. Because these results are not related to any observed change in blood pressure, we believe that naloxone may be achieving its effect through the preservation of spinal cord blood flow, as well as other mechanisms that have yet to be defined.

Electron Paramagnetic Resonance Spectroscopy

The air pollutant ozone Is believed to exert its deleterious biological effects by the formation of free radicals. However these free radicals have not been directly measured. It has also been suggested that peroxidation of cell membrane unsaturated fatty acids is an important mechanism In ozone toxicity. Utilizing electron paramagnetic resonance technique, direct ozonization of linoleic acid produced measurable free radicals after a two-hour induction period.

Molecular Aspects of Membrane Structure in Cerebral Edema

Cerebral edema, regardless of the inciting cause, and irrespective of the cellular sites e.g., tight junctions of vascular structures, glial processes, etc., ultimately represents a pertubation of membrane biomolecules that maintain water and solute compartments. Furthermore, it is likely that in the highly organized tissues of the central nervous system more than one membrane system is involved in the pathogenesis of cerebral edema and the neural dysfunctions that may accompany this process.

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.

Loss of Ascorbic Acid from Injured Feline Spinal Cord

Abstract: Feline spinal cord contains 0.97 mM ascorbic acid, as measured by the dinitrophenylhydrazine method. Greater than 90% is maintained in the reduced form. When functioning normally, the CNS conserves its ascorbic acid with a turnover rate of 2% per h. Following contusion injury severe enough to produce paraplegia, ascorbic acid is rapidly lost from injured spinal tissue. Thus, ascorbic acid is decreased 30% by 1 h and 50% by 3 h following injury. Oxidized ascorbic acid is increased at 1, but not 3, h following impact. As a consequence of its many functions in CNS, loss of ascorbic acid may contribute to derangements in spinal cord function following injury.

PARAMAGNETIC SPECIES AND RADICAL PRODUCTS IN CAT SPINAL CORD

Investigations directed towards understanding some of the biochemical and pathological changes that are initiated as a consequence of spinal cord injury have been pursued at the Milbank Research Laboratories for several In a number of these investigations, cat thoracic spinal cords were exposed by laminectomy and subsequently contused in vivo by applying a reproducible force of impaction.5 Recently, an hypothesis which seeks to explain by biochemical mechanisms the observed pathological effects of trauma has been developed by our group. The hypothesis suggests that lipid peroxidation,6 catalyzed by the hematin compounds derived from extravasated red blood cells in the central gray matter of the contused cord, may be an important factor leading to the ultimate appearance of extensive edema and necrosis in the traumatized cord. The lipid peroxidation chain reaction involving free radical attack by peroxides on the a-methylenic groups in unsaturated fatty acids has been shown to occur in mammalian membranes subjected to physical trauma as well as other pathological procedures.8 Since the cord is extremely rich in lipids (about 40% lipid, 40% cholesterol, and 20% proteins: the unsaturated lipids are localized primarily in the gray matter,g this hypothesis seems attractive. Nerve tissue has been notably lacking in paramagnetic signals, but the recent finding by Commoner and associates lo of possible ferromagnetic centers in mechanically injured sciatic nerve is of considerable interest. An electron spin resonance spectrometer was employed to search for the free radicals or paramagnetic complexes that might be present in the cord as a result of the events involved in lipid peroxidation. Normal noncontused spinal cord was removed from the cat and placed on ice until ready for use. The sample was prepared by placing several thin (-0.5 mm) transverse sections in a Varian quartz tissue cell. A drop of water or isotonic saline was often added as well. The cell was inserted in the cavity of a Varian E-3 spectrometer and measurements were made at an ambient temperature of 27-30°C. No attempt was made either to restrict the samples to white or gray matter alone or to minimize the very small amount of blood that normally is present in the samples. Nevertheless, samples that had apparently significantly different amounts of blood produced the results that are presented below.

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.

The Development of Secondary Pathology with free Radical Reactions as a Threshold Mechanism

Malignancies often develop at sites of ongoing inflammation and chronic ~lcerat ionl-~~. In most of these instances, the inflammation and ulceration are requisite steps in the development of cancer at that particular site. The specific factors, however, that induce such secondary pathology are best not termed carcinogens, but inflammatory agents or ulcerogens that are applied or administered at a dose-rate sufficient to create the secondary pathology. This is therefore a threshold mechanism, i.e., the need to create enough tissue injury to produce inflammatory/ulcerative changes, before a malignancy can develop. The fact that cancers do often develop in conditions of chronic active inflammation and ulceration, as listed in Table 1, suggests two things: