Research reportEffects of tempol, a membrane-permeable radical scavenger, in a gerbil model of brain injury
Introduction
Cerebral ischemia is followed by an inflammatory reaction of the post-ischemic brain [21], [8], [9], [10]. There is evidence that the acute inflammatory reaction associated with reperfusion of the previously ischemic brain contributes to the development of the brain damage (reperfusion injury). Loss of blood flow to the brain results in neuronal injury due to the cessation of blood flow (leading to oxygen and nutrient deprivation) and the injury associated with the reperfusion of the previously ischemic tissue [21]. Thus, both restoration of blood supply and prevention of reperfusion injury are essential to minimize the degree of cerebral infarction caused by transient, cerebral ischemia.
Numerous transmitter and second messenger pathways are inappropriately activated after the initial ischemic event [8]. There is now good evidence that ischemia and reperfusion of the brain leads to a rapid increase in the extracellular levels of glutamate. This results in activation of glutamate receptors, an increase in intracellular calcium, and subsequently in the generation of free radicals and nitric oxide (NO) [10]. Protection against NMDA neurotoxicity occurs following treatment of primary brain cultures with inhibitors of NO synthase (NOS) [11], [17] and cortical cultures from mice with targeted disruption of the neuronal isoform of NOS (nNOS) [16]. The cerebral injury caused by transient cerebral ischemia is also markedly diminished in animals treated with NOS inhibitors [10] or in mice with nNOS gene disruption [16].
Once generated, NO may exert multiple physiological or (when generated in excessive amounts) pathophysiological effects different cell types within the brain. It has been proposed that many of the toxic effects of NO are due to the generation of the potent oxidant peroxynitrite (due to the reaction of NO with superoxide anions) [1], [3]. Peroxynitrite is cytotoxic via a number of independent mechanisms including: (i) the initiation of lipid peroxidation, (ii) the inactivation of a variety of enzymes (most notably, mitochondrial respiratory enzymes and membrane pumps) [25], [18] and (iii) depletion of glutathione [18], [27]. Moreover, peroxynitrite can also cause DNA damage [30], [4], [5], [6] resulting in the activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS). The excessive activation of this enzyme results, in turn, in depletion of NAD and ATP and ultimately cell death [30], [6], [7]. Interventions, which reduce the generation or the effects of peroxynitrite and PARS activation, exert beneficial effects in a variety of models of inflammation, shock and ischemia-reperfusion including the model of cerebral ischemia used in this study. These therapeutic interventions include NOS inhibitor [9], a vitamin E-like antioxidant [2], [6], a SOD-mimetic [29], a peroxynitrite decomposition catalyst [28] and inhibitors of PARS activity [6], [7], [14].
Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), is stable to piperidine nitroxide (stable free radical) of low molecular weight, which permeates biological membranes and scavenges superoxide anions in vitro [19]. It also exerts beneficial effects in rats subjected to endotoxemia [20]. We have recently discovered that tempol reduces the reperfusion injury of rats and rabbits subjected to acute myocardial ischemia and reperfusion [12]. This study investigates the effects of tempol against the oxidative stress, which occurs during the reperfusion of the previously ischemic brain. The following biochemical and morphological parameters were evaluated: (1) Lipid peroxidation, (2) neutrophil accumulation, (3) brain oedema, (4) locomotor hyperactivity, (5) neuronal cell loss, (6) nitration of cerebral proteins, (7) activation of PARS within sections of the brain (immunohistochemistry) and (8) survival.
Section snippets
Animals
Adult male Mongolian gerbils (60–70 g; Charles River; Milan; Italy) were housed in a controlled environment and provided with standard rodent chow and water ad libitum. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purpose (D.M. 116192) as well as with EEC regulations (O.J. of E.C. L 358/1 12/18/1986).
Surgical procedures
General anesthesia was induced with halothane (2%) and maintained with nitrous oxygen (given together with oxygen).
Effect of tempol on the increase in the NO levels caused by BCO
When compared to sham-operated animals, gerbils subjected to cerebral ischemia and reperfusion exhibited a significant increase in the plasma levels of nitrate/nitrite at 4 h after the onset of reperfusion (Fig. 1A). In the hippocampus obtained from animals subjected to BCO, a significant increase of iNOS activity was detected at 4 h (Fig. 1B). Treatment of gerbils with tempol did not affect this increase in the plasma levels of nitrite/nitrate or the increase in iNOS activity in the
Discussion
This study reports for the first time that the membrane-permeable radical scavenger, tempol, reduces the: (i) degree of lipid peroxidation, (ii) brain edema, (iii) accumulation of neutrophils (cortex and hippocampus), (iv) locomotor hyperactivity, (v) hippocampal neuronal loss and (vi) mortality caused by transient, cerebral ischemia and reperfusion in the gerbil.
We propose that the beneficial effects of tempol on brain injury is due to the ability of this stable free nitroxide radical to
Acknowledgements
This work was supported by Ministero Pubblica Istruzione, Fondi 40%. The authors would like to thank Giovanni Pergolizzi and Carmelo La Spada for their excellent technical assistance during this study, Mrs. Caterina Cutrona for secretarial assistance and Miss Valentina Malvagni for editorial assistance with the manuscript. C.T. is a Senior Fellow of the British Heart Foundation (FS 96/018).
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