Paul Demediuk

Effects of Methylprednisolone and the Combination of α-Tocopherol and Selenium on Arachidonic Acid Metabolism and Lipid Peroxidation in Traumatized Spinal Cord Tissue

Abstract: Traumatic injury of the spinal cord leads to a series of pathological events that result in tissue necrosis and paralysis. Among the earliest biochemical reactions are hydrolysis of fatty acids from membrane phospholipids, production of biologically active eicosanoids, and peroxidation of lipids. This study examines the effect of agents purported to improve recovery following spinal cord trauma, methyl‐prednisolone sodium succinate (MPSS) and the combination of α‐tocopherol and selenium (Se), on the posttraumatic alterations of membrane lipid metabolism. Pretreatment with either MPSS or a‐tocopherol and Se reduced the trauma‐induced release of total FFA including arachidonate in the injured spinal cord tissue. In addition, these agents decreased the postinjury levels of prostanoids. Pre‐treatment with either MPSS or a‐tocopherol and Se also completely prevented the trauma‐induced loss of cholesterol while inhibiting the increase of a cholesterol peroxidation product, 25‐hydroxycholesterol. These data suggest that: (a) perturbation of membrane lipid metabolism may contribute to the tissue necrosis and functional deficit of spinal cord injury and (b) MPSS or the combination of a‐tocopherol and Se may protect injured spinal cord tissue, at least in part, by limiting these posttraumatic membrane lipid changes.

Spinal cord injury and protection

Subsequent to traumatic injury of the spinal cord, a series of pathophysiological events occurs in the injured tissue that leads to tissue destruction and paraplegia. These include hemorrhagic necrosis, ischemia, edema, inflammation, neuronophagia, loss of Ca2+ from the extracellular space, and loss of K+ from the intracellular space. In addition, there is trauma-initiated lipid peroxidation and hydrolysis in cellular membranes. Both lipid peroxidation and hydrolysis can damage cells directly; hydrolysis also results in the formation of the biologically active prostaglandins and leukotrienes (eicosanoids). The time course of membrane lipid alterations seen in studies of antioxidant interventions suggests that posttraumatic ischemia, edema, inflammation, and ionic fluxes are the result of extensive membrane peroxidative reactions and lipolysis that produce vasoactive and chemotactic eicosanoids. A diverse group of compounds has been shown to be effective in ameliorating spinal cord injury in experimental animals. These include the synthetic glucocorticoid methylprednisolone sodium succinate (MPSS); the antioxidants vitamin E, selenium, and dimethyl sulfoxide (DMSO); the opiate antagonist naloxone; and thyrotropin-releasing hormone (TRH). With the exception of TRH, all of these agents have demonstrable antioxidant and/or anti-lipid-hydrolysis properties. Thus the effectiveness of these substances may lie in their ability to quench membrane peroxidative reactions or to inhibit the release of fatty acids from membrane phospholipids, or both. Whatever the mode of action, early administration appears to be a requirement for maximum effectiveness.

Separation of phospholipids by high-performance liquid chromatography: all major classes, including ethanolamine and choline plasmalogens, and most minor classes, including lysophosphatidylethanolamine

High-performance liquid chromatographic methods for the separation and quantitation of phospholipids were developed and shown to give sensitive, reliable measurements of tissue phospholipids, including difficult-to-resolve pairs such as choline plasmalogen (plasmenylcholine) and phosphatidylcholine, choline glycerophospholipids and sphingomyelin, phosphatidylinositol and phosphatidylserine, and phosphatidylserine and lysophosphatidylethanolamine. Separations of most phospholipids including those mentioned above are more complete than in existing procedures, and require only 40 min per injection. Utilization of the hexane-2-propanol-water system has an advantage over separation techniques that employ acidic solvents in that the plasmalogens are not hydrolyzed and a less degradative environment for labile lipids is provided. Further, a rapid high-performance liquid chromatographic procedure for the separation of intact ethanolamine plasmalogen (plasmenylethanolamine) from phosphatidylethanolamine was developed. Previous procedures have required derivatized samples or acid hydrolysis of the plasmalogen vinyl ether linkage. A slight modification of the primary method (method I) increases the resolution of lysophosphatidylethanolamine from other classes (method II). A third modification (method III) can replace the standard silicic acid column separation of lipids into neutral, glycolipid, and phospholipid fractions.

Mechanical damage to murine neuronal-enriched cultures during harvesting: Effects of free fatty acids, diglycerides, Na+, K+-ATPase, and lipid peroxidation

SummaryThe most commonly used procedure to harvest cultured cells from petri dishes is to scrape the cells off the plates with a rubber or Teflon policeman. However, the results reported herein demonstrate that this technique, with its associated mechanical trauma, significantly perturbed cell membranes in neuronal-enriched cultures derived from the ventral half of fetal murine spinal cords. This is evidenced by liberation of free fatty acids and diglycerides, partial inhibition of Na+K+-ATPase activity, and increased malondialdehyde production. Harvesting the cells by freezing, either on liquid nitrogen or dry ice, significantly attenuated these effects. This important observation indicates that mechanical manipulation of cultured cells during harvesting significantly affects subsequent biochemical analyses, particularly those associated with the cell membrane (e.g., membrane lipid metabolism and assay of intrinsic membrane enzymes).

Early membrane lipid changes in laminectomized and traumatized cat spinal cord.

The effects of surgical exposure (laminectomy) and compression trauma on various aspects of membrane lipid metabolism in the feline spinal cord were determined in this study. Tissue samples were frozen in situ and grossly dissected into gray and white portions prior to lipid analyses. Laminectomy alone resulted in measurable changes in spinal cord lipid metabolism, including increases in gray matter free fatty acids, diacylglycerols, and eicosanoids. A 90-min recovery period greatly reduced the levels of these compounds. Compression of the spinal cord with a 170-g weight (following a 90-min recovery period) caused very large increases in gray matter free fatty acids, diacylglycerols, and eicosanoids, and decreases in cholesterol and ethanolamine plasmalogens. Similar, but time delayed changes in these compounds were also observed in white matter.

Neurobehavioral Effects of Chronic Choline-Containing Diets on the Adult and Aging C57BL/6NNIA Mouse Braina

Choline-containing diets were chronically fed to male C57BL/6NNIA mice for either 5 or 11 months: from 8-13 months or 13-24 months, respectively. The choline in the chow was supplied in one of three ways: as free choline (choline chloride) or as bound choline as found in a 95% purified preparation of phosphatidylcholine (PC) and in an oil-free granular lecithin formulation (centrolex). The choline in these diets (either free or bound forms) was enriched at low, medium, or high levels (containing 2.4, 4.8, or 10.8 mg/g of chow, respectively). Two low choline diets contained 0.9 and 1.5 mg/g of choline, respectively, but were regarded as choline-adequate since the minimal nutritional requirement for choline is thought to be 0.6 mg/g of chow. All these diets were isocaloric and isonitrogenous. A standard rodent laboratory chow (Purina) contained 2.3 mg/g of choline. One-trial passive-avoidance testing for retention of learning indicated that mice of the C57BL/6NNIA strain show little normal age-related memory loss between 8-24 months old. As such, dietary enrichments did not significantly improve performance in comparison to the mice on the standard lab chow containing abundant choline. Learning was improved, however, in relation to mice on lower choline control diets, by supplementation with choline, PC, or lecithin. Whereas the younger mice tended to respond better a t higher levels of enrichment, the older (24 months old) mice showed superior retention of learning following low enrichment levels of PC and lecithin. This suggests that there may be an age-related shift in the optimal, potentially prophylactic, dietary “window.” Additional studies are evaluating some parameters reflecting potential membrane changes as a consequence of the various dietary regimens. Analysis of membrane phospholipids in a plasma membrane fraction from mouse forebrain indicated that membrane composition remains remarkably constant; however, diet-modulated enhanced membrane fluidity is suggested by a reduced cholesterol-to-phospholipid ratio in the older mice on low levels of chronic dietary enrichment. Receptor-binding studies from the neocortex (muscarinic, aand 8-adrenergic), hippocampus (musca-

Effect of dietary choline on membrane composition of brain in the aging mouse

Sympathetic denervation of rat heart by administration of 6-hydroxydopamine (6OHDA) results in an increase in the activity of factors required for the survival of 12 day old chick embryo lumbar sympathetic ganglien neurones in dissociated cell culture. A large proportion of this increased activity can be blocked with affinity purified antibodies to mouse salivary gland nerve growth factor CNGF). In order to further investigate this increase we have fractionsted extracts of normal and denervated heart using both gel filtration on sephadex G-100 and immuno-affinity chromatography with antibodies to mouse NGF linked to sepharose. In control heart there are two components with apparent molecular weight on sephadex G-100 of 40,000 and 12,000 that have sympathetic survival activity but neither of these is NGF. After denervation, NGF can be detected at a level of 1.6 ng/gm in the heart and comprises the major component of the increased survival activity. In addition the two other components are increased in activity by 200% and 4504 respectively. ThSs suggests that all three components are regulated by innervation either by a direct action on their synthesis or by removal due to retrograde axonal transport. Alternatively NGF is only important during initial development and regeneration while the other two components are more important for the on-going maintenance of sympathetic neurones.

Membrane lipids and aging

Abstract The concept that age-related changes in lipids of brain membranes can “damage” membranes through the loss of communicative coupling of bilayer halves of biomembranes is critiqued and expanded to include the possibility that changes in the asymmetrically distributed gangliosides, in ethanolamine plasmalogens, and in cholesterol may also contribute to such damage. The possibility for attenuation or reversal of lipid changes by dietary manipulations or treatment with liposomes composed of phosphatidylserine is also briefly introduced.