The HPLC method used in this study and previously with a neural cell model of chemical hypoxia28 has several advantages over other commonly used assays of lipid oxidation. This method directly measures the primary products of lipid peroxidation, ie, lipid hydroperoxides and hydroxides, rather than secondary, lower-molecular-weight breakdown products, eg, malondialdehyde, ethane, or pentane. The use of HPLC to separate 235-nm absorbing species also greatly minimizes interference by molecules other than oxidized lipids that also absorb light of this wavelength and can contaminate simple Folch tissue extracts. Other investigators have also shown HPLC separation of oxidized lipids together with alternative methods of detection, eg, chemiluminescence, to be a sensitive and reliable method of quantifying oxidized lipids present in tissue extracts.31 32 In addition to using an HPLC procedure to quantify the level of total oxidized fatty acyl groups present in brain biopsies, the present study also used gas chromatography/mass spectrometry to actually identify the primary species of oxidized lipid. These species were identified as 13- and 9-HODE, each being oxidation products of linoleic acid. As the Folch brain lipid extracts were reduced with triphenylphosphine to improve stability, the true products formed in vivo may actually be the respective hydroperoxides. Although these products can be derived from lipoxygenase- or cytochrome P-450–dependent enzymatic peroxidation,33 34 they can also arise from nonenzymatic oxidation via attack of fatty acyl groups by hydroxyl radicals or other free radicals thought to be generated at abnormally high levels during ischemia/reperfusion. The significance of the generation of these specific oxidized fatty acids during cerebral ischemia and reperfusion is, at this juncture, speculative. However, oxidation of phospholipid linoleic acid has been demonstrated to be cytotoxic15 35 and has been associated with oxidative DNA damage.14 13- and 9-HODE also exhibit biological activities strongly implicated in ischemic brain injury, eg, chemotactic activity for polymorphonuclear leukocytes36 and the ability to activate cellular protein kinase C.37 Although 13- and 9-HODE were consistently the most predominant oxidized lipids found among different animal groups and throughout different areas of the brain, they only represented 20% to 35% of the area typically observed for at least 10 different 235-nm absorbing peaks eluted from the HPLC column. Further effort is being made to identify these other species of oxidized lipid. The increase in oxidized brain lipids observed after global cerebral ischemia and reperfusion in this study is consistent with the results of other studies with other animal models1 2 3 5 6 7 8 9 10 11 16 and consistent with our previous results with the canine model in which cerebral cortex protein oxidation was demonstrated.21 In contrast to our observations for protein oxidation, the level of oxidized lipids did not increase with increasing periods of reperfusion (Figure 2⇑). This finding suggests that oxidized fatty acyl groups do not accumulate but rather exist in a dynamic state in which degradation to smaller products, eg, malondialdehyde, is balanced by ongoing production of new lipid peroxides and hydroxides. One of the most intriguing observations made in this study was that significant lipid oxidation occurred during the 10-minute period of ischemia in the absence of reperfusion. The fact that this result was obtained with a cardiac arrest model of global cerebral ischemia is significant since a complete lack of blood flow to all parts of the brain unquestionably occurs within seconds after the induction of ventricular fibrillation, whereas some flow of blood to various regions of the brain can occur in many vascular occlusion models of “complete” cerebral ischemia,38 including those where electron spin resonance/spin trapping measurements have indicated the formation of free radicals during the period of ischemia.39 40 Although direct evidence of lipid oxidation during complete cerebral ischemia is scarce, elevated levels of malondialdehyde have been reported in forebrain mitochondria after 30 minutes of ischemia in a standard rat 4-vessel occlusion model41 and in canine parietal cortex after 15 minutes of cardiac arrest.10 Taken together the results of these studies suggest that free radical–induced molecular alterations may contribute to tissue injury during complete ischemia and during various phases of reperfusion. Although significant brain lipid oxidation can occur following ischemia alone, the fact that it is an ongoing, dynamic process makes this form of molecular injury susceptible to postischemic intervention. The present study provides new, direct evidence that hyperoxic resuscitation and reperfusion exacerbates postischemic lipid oxidation. Under these conditions a significant increase in lipid oxidation occurred in the striatum and hippocampus as well as in the frontal cortex. Although the degree of reperfusion-dependent lipid oxidation was not significantly different among these areas, the observation of a trend toward the greatest increase in the striatum is consistent with the findings of Zhang et al32 that indicated that the increase in phospholipid hydroperoxides during aging in gerbils is greatest in the striatum. Zwemer et al24 previously provided evidence with a similar canine cardiac arrest model that hyperoxic, postischemic ventilation results in significantly worse neurological outcome when compared with normoxic ventilation (21% O2). The finding that pretreatment of animals in their hyperoxic group with the antioxidant tirilizad mesylate improved neurological outcome suggested that increased oxidative molecular alterations, eg, lipid peroxidation, may contribute to the deleterious effects of hyperoxic ventilation; however, no direct measurements of such alterations were provided. In another recent study in which an intracranial fluid compression model of global cerebral ischemia in rabbits was used, immediate postischemic treatment with hyperbaric oxygen appeared to increase the production of free radicals, as reflected by an increase in the ratio of brain oxidized/reduced glutathione; however, no increase in the oxidized lipid breakdown product malondialdehyde was observed after 75 minutes of reperfusion.42 These findings suggest that either immediate postischemic hyperbaric oxygen is not as neurotoxic as normobaric hyperoxygenation or that brain malondialdehyde measured within the first 1 to 2 hours of reperfusion is not as sensitive an indicator of lipid oxidation as our HPLC measurements of discrete 235-nm absorbing species of fatty acyl groups performed after 24 hours of reperfusion. The results of the present study not only confirm that normobaric, hyperoxic postcardiac arrest ventilation can be neurologically detrimental but they also demonstrate a close, albeit correlative, relationship between increased neurological impairment and increased frontal cortex lipid oxidation. The additional present finding that hyperoxic reperfusion actually exacerbates rather than ameliorates brain lactic acidosis also challenges the notion that the prolonged use of 100% ventilatory O2 after cardiac arrest may be beneficial through stimulation of aerobic and inhibition of anaerobic cerebral energy metabolism. The neurological and neurochemical results of this study taken together with those of the study by Mickel et al22 and the neurological results of Zwemer et al24 cast serious doubt on the appropriateness of the present Advanced Cardiac Life Support guidelines that recommend the use of 100% ventilatory O2 for undefined periods during and after resuscitation from cardiac arrest.23 However, the present study used only one 10-minute period of cardiac arrest in young healthy animals. Humans are resuscitated after widely variable periods of cardiac arrest and are often elderly with impaired respiratory and cardiovascular systems. Clearly, clinical trials will be necessary to resolve this issue.
Posted on: Mon, 08 Sep 2014 10:06:30 +0000
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