Background. The mechanisms involving the initiation of apoptosis after brain hypoxia-ischemia through caspase activation are not fully defined. Oxygen free radicals may be an important mediator of caspase initiation with reactive oxygen species generated by xanthine oxidase (XO) being one potential source. The purpose of this study was to examine the role of XO in apoptosis after global cerebral injury. Methods. Immature rabbits were subjected to 8 minutes hypoxia and 8 minutes ischemia and then 4 hours of reperfusion. In one group ( ), the XO substrate xanthine was infused to generate more oxygen free radicals to promote apoptosis while in another ( ), the XO inhibitor allopurinol was given to reduce apoptosis by preventing free radical production ( ). Control animals ( ) received the vehicles. Caspase 3, 8, and 9 enzyme activities were measured in the cerebral cortex, hippocampus, cerebellum, thalamus, and caudate. Results. Administration of xanthine increased ( ) caspase 3 activity but only in the hippocampus, and pretreatment with allopurinol did not reduce it. No differences ( ) were found in any other region nor were there any changes in caspases 8 or 9 activities. Conclusion. We conclude that XO is not a major factor in inducing apoptosis after hypoxic-ischemic brain injury. 1. Introduction The reperfusion of previously ischemic tissue is believed to contribute to injury through the generation of free radical species [1, 2]. During hypoxia, ATP is depleted and metabolized through various intermediates to hypoxanthine. When perfusion is reestablished, newly provided oxygen allows for the conversion of hypoxanthine to xanthine, and ultimately to uric acid, by the enzyme xanthine oxidase (XO) [1]. This metabolism of hypoxanthine and xanthine to uric acid by XO has been argued to be a source of oxygen free radicals causing cerebral injury [2–5]. However, in various animal models, inhibition of XO with allopurinol before ischemia has yielded inconclusive results [6–11]. Hypoxia-ischemia leads to not only necrosis but also apoptosis. Apoptosis is a highly ordered process of programmed cell death that can occur in both normal physiologic remodeling and pathologic processes [12]. This type of cell death can be recognized by several biochemical and morphological markers, such as chromatin condensation, DNA fragmentation, caspase activation, and mitochondrial alterations. Apoptosis may be the primary factor in the prolonged progression of neurodegeneration and cerebral dysfunction hours to days after injury [12, 13]. Evidence suggests that apoptosis after
References
[1]
D. N. Granger, M. E. Hollwarth, and D. A. Parks, “Ischemia-reperfusion injury: role of oxygen-derived free radicals,” Acta Physiologica Scandinavica, vol. 126, supplement 548, pp. 47–63, 1986.
[2]
P. Pasdois, J. E. Parker, E. J. Griffiths, and A. P. Halestrap, “The role of oxidized cytochrome c in regulating mitochondrial reactive oxygen species production and its perturbation in ischaemia,” Biochemical Journal, vol. 436, no. 2, pp. 493–505, 2011.
[3]
T. Cherin, M. Catbagan, S. Treiman, and R. Mink, “The effect of normothermic and hypothermic hypoxia-ischemia on brain hypoxanthine phosphoribosyl transferase activity,” Neurological Research, vol. 28, no. 8, pp. 831–836, 2006.
[4]
T. D. Engerson, G. McKelvey, and D. B. Rhyne, “Conversion of xanthine dehydrogenase to oxidase in ischemic rat tissues,” Journal of Clinical Investigation, vol. 79, no. 6, pp. 1564–1570, 1987.
[5]
A. Valencia and J. Morán, “Reactive oxygen species induce different cell death mechanisms in cultured neurons,” Free Radical Biology and Medicine, vol. 36, no. 9, pp. 1112–1125, 2004.
[6]
T. Itoh, M. Kawakami, and Y. Yamauchi, “Effect of allopurinol on ischemia and reperfusion-induced cerebral injury in spontaneously hypertensive rats,” Stroke, vol. 17, no. 6, pp. 1284–1287, 1986.
[7]
D. Martz, G. Rayos, G. P. Schielke, and A. L. Betz, “Allopurinol and dimethylthiourea reduce brain infarction following middle cerebral artery occlusion in rats,” Stroke, vol. 20, no. 4, pp. 488–494, 1989.
[8]
R. Mink and J. Johnston, “The effect of infusing hypoxanthine or xanthine on hypoxic-ischemic brain injury in rabbits,” Brain Research, vol. 1147, no. 1, pp. 256–264, 2007.
[9]
R. B. Mink, A. J. Dutka, and J. M. Hallenbeck, “Allopurinol pretreatment improves evoked response recovery following global cerebral ischemia in dogs,” Stroke, vol. 22, no. 5, pp. 660–665, 1991.
[10]
R. B. Mink, A. J. Dutka, K. K. Kumaroo, and J. M. Hallenbeck, “No conversion of xanthine dehydrogenase to oxidase in canine cerebral ischemia,” American Journal of Physiology, vol. 259, no. 6, pp. H1655–H1659, 1990.
[11]
L. S. Terada, J. D. Rubinstein, E. J. Lesnefsky, L. D. Horwitz, J. A. Leff, and J. E. Repine, “Existence and participation of xanthine oxidase in reperfusion injury of ischemic rabbit myocardium,” American Journal of Physiology, vol. 260, no. 3, pp. H805–H810, 1991.
[12]
B. W. B？ttiger, B. Schmitz, C. Wiessner, P. Vogel, and K. Hossmann, “Neuronal stress response and neuronal cell damage after cardiocirculatory arrest in rats,” Journal of Cerebral Blood Flow and Metabolism, vol. 18, no. 10, pp. 1077–1087, 1998.
[13]
O. P. Mishra and M. Delivoria-Papadopoulos, “Effect of neuronal nitric oxide synthase inhibition on caspase-9 activity during hypoxia in the cerebral cortex of newborn piglets,” Neuroscience Letters, vol. 401, no. 1-2, pp. 81–85, 2006.
[14]
J. M. McCord, “Oxygen-derived free radicals in postischemic tissue injury,” New England Journal of Medicine, vol. 312, no. 3, pp. 159–163, 1985.
[15]
A. Sola, V. Alfaro, and G. Hotter, “Intestinal ischemic preconditioning: less xanthine accumulation relates with less apoptosis,” Apoptosis, vol. 9, no. 3, pp. 353–361, 2004.
[16]
Y. Cheng, M. Deshumukh, A. D. Costa, et al., “Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury,” Journal of Clinical Investigation, vol. 101, pp. 1992–1999, 1998.
[17]
K. A. Elliott and H. H. Jasper, “Physiological salt solutions for brain surgery; studies of local pH and,” Journal of Neurosurgery, vol. 6, no. 2, pp. 140–152, 1949.
[18]
S. Treiman, M. Catbagan, C. Hatchette, T. Cherin, and R. Mink, “Administration of allopurinol and hypoxanthine improves recovery of adenine nucleotides after cerebral hypoxia-ischemia in rabbits,” Critical Care Medicine, vol. 31, no. 2, p. A24, 2002.
[19]
C. Peeters-Scholte, K. Braun, J. Koster et al., “Effects of allopurinol and deferoxamine on reperfusion injury of the brain in newborn piglets after neonatal hypoxia-ischemia,” Pediatric Research, vol. 54, no. 4, pp. 516–522, 2003.
[20]
R. B？genholm, U. A. Nilsson, C. W. G？tborg, and I. Kjellmer, “Free radicals are formed in the brain of fetal sheep during reperfusion after cerebral ischemia,” Pediatric Research, vol. 43, no. 2, pp. 271–275, 1998.
[21]
H. A. Kontos, “Oxygen radicals in cerebral ischemia: the 2001 Willis lecture,” Stroke, vol. 32, no. 11, pp. 2712–2716, 2001.
[22]
B. Herrera, M. M. Murillo, A. álvarez-Barrientos, J. Beltrán, M. Fernández, and I. Fabregat, “Source of early reactive oxygen species in the apoptosis induced by transforming growth factor-β in fetal rat hepatocytes,” Free Radical Biology and Medicine, vol. 36, no. 1, pp. 16–26, 2004.
[23]
P. Khurana, Q. M. Ashraf, O. P. Mishra, and M. Delivoria-Papadopoulos, “Effect of hypoxia on caspase-3, -8, and -9 activity and expression in the cerebral cortex of newborn piglets,” Neurochemical Research, vol. 27, no. 9, pp. 931–938, 2002.
[24]
A. Pirzadeh, A. Mammen, J. Kubin et al., “Early regional response of apoptotic activity in newborn piglet brain following hypoxia and ischemia,” Neurochemical Research, vol. 36, no. 1, pp. 83–92, 2011.
[25]
H. Tamta, S. Kalra, and A. K. Mukhopadhyay, “Biochemical characterization of some pyrazolopyrimidine-based inhibitors of xanthine oxidase,” Biochemistry, vol. 71, supplement 1, pp. S49–S54, 2006.
[26]
J. Y. Wang, S. C. Sorice, and R. B. Mink, “The effect of allopurinol on guanine-based purine metabolism after cerebral hypoxia-ischemia in rabbits,” Critical Care Medicine, vol. 34, p. A37, 2006.
[27]
S. Pirouzpanah, J. Hanaee, S. Razavieh, and M. Rashidi, “Inhibitory effects of flavonoids on aldehyde oxidase activity,” Journal of Enzyme Inhibition and Medicinal Chemistry, vol. 24, no. 1, pp. 14–21, 2009.
[28]
L. Joly, V. Mucignat, J. Mariani, M. Plotkine, and C. Charriaut-Marlangue, “Caspase inhibition after neonatal ischemia in the rat brain,” Journal of Cerebral Blood Flow and Metabolism, vol. 24, no. 1, pp. 124–131, 2004.
