NM  Vol.1 No.2 , December 2010
Added after Anoxia-Reoxigenation Stress, Genistein Rescues from Death the Rat Embryo Cortical Neurons
ABSTRACT
Estrogens and phytoestrogens have neuroprotective effect against neuronal damage induced by cerebral ischemia /reperfusion (I/R) injury. In preceding studies, the phytoestrogen effects have been assessed by administration previous to the ischemic period, conditions which are unusual to apply to the treatment of human stroke. Here we present a study on neuroprotection afforded by genistein on rat embryo cortical neurons subjected to oxygen and glucose deprivation (OGD) followed by re-oxigenation, when added after the stress stimulus. At 1 and 2 h of OGD times and after 24 h of reperfusion, cell viability, necrotic, apoptotic and autophagic cell death and different parameters related to oxidative stress and mitochondrial dysfunction were measured in the absence and presence of 1 µM genisteine. We found an in-creasing loss of neuronal viability after 1-5 h of OGD which was only reversed in part by 24 h of reperfusion. These changes were preceded by increases in ROS generation, caspase-3 activation, LDH release and increase in LC3B lipi-dation, indicative of autophagia. Treatment with 1 µM genistein during the 24 h reperfusion significantly attenuated neuronal necrosis and autophagia induced by 1 and 2 h of OGD exposure. Genistein also decreased ROS generation and lipid-peroxidation induced by 2 h of OGD. These results suggest an important neuroprotective effect of genistein against transient post-ischemic-like conditions

Cite this paper
nullA. Carmen, A. José Luis, S. Eduardo, O. Ma Jesús and G. Ma Pilar, "Added after Anoxia-Reoxigenation Stress, Genistein Rescues from Death the Rat Embryo Cortical Neurons," Neuroscience and Medicine, Vol. 1 No. 2, 2010, pp. 50-59. doi: 10.4236/nm.2010.12008.
References
[1]   M. P. Mattson and R. J. Mark. “Excitotoxicity and Excitoprotection in Vitro,” Advances in Neurology, Vol.71, 1996, pp 1-30.

[2]   U. Dirnagl, C. Iadecola, and M. A. Moskowitz, “Pathobiology of Ischemic Stroke: An Integrated View,” Trends Neuroscience, Vol. 22, No. 9, 1999, pp 391-397. doi:10.1016/S0166-2236(99)01401-0

[3]   L. A. Labiche and J. C. Grotta, “Clinical Trials for Cytoprotection in Stroke,” NeuroRx, Vol. 1, No. 1, 2004, pp. 46-70. doi:10.1602/neurorx.1.1.46

[4]   M. A. de Leci?ana and J. A. Egido, “Estrogens as Neuroprotectants against Ischemic Stroke,” Cerebrovascular Diseases, Vol. 21 No. 2, 2006, pp. 48-53. doi:10.1159/000091703

[5]   S. Suzuki, C. M. Brown and P. M. Wise, “Neuroprotective Effects of Estrogens Following Ischemic Stroke,” Frontiers in Neuroendocrinology, Vol. 30, No. 2, 2009, pp. 201-211. doi:10.1016/j.yfrne.2009.04.007

[6]   C. L. Gibson, L. J. Gray, S. P. Murphy and P. M. Bath, “Estrogens and Experimental Ischemic Stroke: A Systematic Review,” Journal of Cerebral Blood Flow & Metabolism, Vol. 26, No. 9, 2006, pp. 1103-1113.

[7]   T. Jover, H. Tanaka, A. Calderone, K. Oguro, M. V. Bennett, A. M. Etgen and R. S. Zukin, “Estrogen Protects against Global Ischemia-Induced Neuronal Death and Prevents Activation of Apoptotic Signaling Cascades in the Hippocampal Ca1,” Journal of Neuroscience, Vol. 22, No. 6, 2002, pp. 2115-2124.

[8]   C. A. Singer, X. A. Figueroa-Masot, R. H. Batchelor and D. M. Dorsa. “The Mitogen-Activated Protein Kinase Pathway Mediates Estrogen Neuroprotection after Glutamate Toxicity in Primary Cortical Neurons,” Journal of Neuroscience, Vol. 19, No. 7, 1999, pp. 2455-2463.

[9]   K. Honda, H. Sawada, T. Kihara, M. Urushitani, T. Nakamizo, A. Akaike and S. Shimohama, “Phosphatidylinositol 3-Kinase Mediates Neuroprotection by Estrogen in Cultured Cortical Neurons,” Journal of Neuroscience Research, Vol. 60, No. 3, 2000, pp. 321-327. doi:10.1002/(SICI)1097-4547(20000501)60:3<321::AID-JNR6>3.0.CO;2-T

[10]   E. A. Sribnick, S. K. Ray, M. W. Nowak, L. Li and N. L. Banik, “17 Beta-Estradiol Attenuates Glutamate-Induced Apoptosis and Preserves Electrophysiologic Function in Primary Cortical Neurons,” Journal of Neuroscience Research, Vol. 76, No. 5, 2004, pp. 688-696. doi:10.1002/jnr.20124

[11]   J. Nilsen, and R. D. Brinton, “Impact of Progestins on Estrogen-Induced Neuroprotection: Synergy by Progesterone and 19-Norprogesterone and Antagonism by Medroxyprogesterone Acetate,” Endocrinology, Vol. 13, No. 6, 2002, pp. 205-212. doi:10.1210/en.143.1.205

[12]   H. Cimarosti, I. R.Siqueira, L. L. Zamin, M. Nassif, R. Balk, R. Frozza, C. Dalmaz, C. A. Netto and C. Salbego, “Neuroprotection and Protein Damage Prevention by Estradiol Replacement in Rat Hippocampal Slices Exposed to Oxygen-Glucose Deprivation,” Neurochemical Research, Vol. 30, 2005, pp. 583-589. doi:10.1007/s11064-005-2693-1

[13]   S. Chen, J. Nilsen and R. D. Brinton, “Dose and Temporal Pattern of Estrogen Exposure Determines Neuroprotective Outcome in Hippocampal Neurons: Therapeutic Implications,” Endocrinology, Vol. 147, No. 11, 2006, pp. 5303-5313. doi:10.1210/en.2006-0495

[14]   E. Candelario-Jalil, N. H. Mhadu, S. M. Al-Dalain, G. Martinez and O. S. León, “Time Course of Oxidative Damage in Differenr Brain Regions Following Transient Cerebral Ischemia in Gerbis,” Neurochemical Research, Vol. 41, No. 3, 2001, pp. 233-241.

[15]   D. Amantea, R. Russo, G. Bagetta and M. T.Corasaniti. “From Clinical Evidence to Molecular Mechanisms Underlying Neuroprotection Afforded by Estrogens,” Pharmacol Research, Vol. 52, No. 2, 2005, pp. 119-132. doi:10.1016/j.phrs.2005.03.002

[16]   V. N. Trieu and F. M. Uckun, “Genistein is Neuroprotective in Murine Models of Familial Amyotrophic Lateral Sclerosis and Stroke,” Biochemical and Biophysical Research Communications, Vol. 258, No. 3, 1999, pp 685- 688. doi:10.1006/bbrc.1999.0577

[17]   D. A. Schreihofer and L. Redmond. “Soy Phytoestrogens are Neuroprotective against Stroke-Like Injury in Vitro,” Neuroscience, Vol. 158, No. 2, 2009, pp 602-609. doi:10.1016/j.neuroscience.2008.10.003

[18]   M. C. Burguete, G. Torregrosa, F. J. Perez-Asensio, M. Castello-Ruiz, J. B. Salom, J. V. Gil and E. Alborch, “Dietary Phytoestrogens Improve Stroke Outcome after Transient Focal Cerebral Ischemia in Rats,” European Journal of Neuroscience, Vol. 23, No. 2, 2006, pp. 703-710. doi:10.1111/j.1460-9568.2006.04599.x

[19]   L. Zhao, Q. Chen and R. D. Brinton, “Neuroprotective and Neurotrophic Efficacy of Phytoestrogens in Cultured Hippocampal Neurons,” Experimental Biology and Medicine, Vol. 227, No. 7, 2002, pp. 509-519.

[20]   N. J. Linford and D. M Dorsa, “17 Beta-Estradiol and the Phytoestrogen Genistein Attenuate Neuronal Apoptosis Induced by the Endoplasmic Reticulum Calcium-Atpase Inhibitor Thapsigargin,” Steroids, Vol. 67, No. 13-14, 2002, pp. 1029-1040. doi:10.1016/S0039-128X(02)00062-4

[21]   H. Zeng, Q. Chen and B. Zhao, “Genistein Ameliorates Beta- Amyloid Peptide (25-35)-Induced Hippocampal Neuronal Apoptosis,” Free Radical Biology and Medicine, Vol. 36, No. 2, 2004, pp. 180-188. doi:10.1016/j.freeradbiomed.2003.10.018

[22]   H. Inoue, H. Ohtaki, T. Nakamachi, S. Shioda and Y. Okada, “Anion Channel Blockers Attenuated Delayed Neuronal Cell Death Induced by Transient Forebrain Ischemia,” Journal of Neuroscience Research, Vol. 85, No. 7, 2007, pp. 1427-1435. doi:10.1002/jnr.21279

[23]   K. P. Ho, L. Li, L. Zhao and Z. M. Qjan, “Genistein Protects Primary Cortical Neurons from Iron Induced Lipid Peroxidation,” Molecular and Cellular Biochemistry, Vol. 247, No. 1-2, 2003, pp. 219-222. doi:10.1023/A:1024142004575

[24]   H. W. Liang, S. F. Qju, J. Shen, L. N. Sun, J. Y. Wang, I. C. Bruce and Q. Xia, “Genistein Attenuates Oxidative Stress and Neuronal Damage Following Transient Global Cerebral Ischemia in Rat Hippocampus,” Neuroscience Letters, Vol. 438. No. 1, 2008, pp. 116-120. doi:10.1016/j.neulet.2008.04.058

[25]   S. Figueroa, M. J. Oset-Gasque, C. Arce, C. Martínez- Honduvilla and M. P. González, “Mitochondrial Involvement in Nitric Oxide-Induced Cellular Death in Cortical Neurons in Culture,” Journal of Neuroscience Research, Vol. 83, No. 3, 2006, pp. 441-449. doi:10.1002/jnr.20739

[26]   M. Bradford, “A Rapid Sensitive Method for the Quantization of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding,” Analytical Biochemistry, Vol. 72, 1976, pp. 248-254. doi:10.1016/0003-2697(76)90527-3

[27]   L. Tenneti, D. M. D'Emilia, C. M. Troy and S.A Lipton, “Role of Caspases in N-Methyl-D-Aspartate-Induced Apoptosis in Cerebrocortical Neurons,” Journal of Neurochemistry, Vol. 71, No. 3, 1998, pp. 946-959. doi:10.1046/j.1471-4159.1998.71030946.x

[28]   A. S. Waggoner. “The Use of Cyanine Dyes for the Determination of Membrane Potentials in Cells, Organelles, and Vesicles,” Methods in Enzymology, Vol. 55, 1979, pp 689-695. doi:10.1016/0076-6879(79)55077-0

[29]   C. G. Fraga, P. I. Oteiza, M. S. Golup, M. E. Gershwin and C. L. Keen, “Effects of Aluminium on Brain Lipid Peroxidation,” Toxicology Letters, Vol. 51, No. 2, 1990, pp. 213-219. doi:10.1016/0378-4274(90)90212-5

[30]   L. Xue, G. C. Fletcher and A. M. Tolkovsky, “Autophagy is Activated by Apoptotic Signaling in Sympathetic Neurons: An Alternative Mechanism of Death Execution,” Molecular and Cellular Neuroscience, Vol. 14, No. 3, 1999, pp. 180-198. doi:10.1006/mcne.1999.0780

[31]   P. Codogno and A. J. Mejier, “Autophagy and Signalling: Their Role in Cell Survival and Cell Death,” Cell Death and Differentiation, Vol. 12 No. S2, 2005, pp. 1509-1518. doi:10.1038/sj.cdd.4401751

[32]   F. Adhami, G. Liao, Y. M. Morozov, A. Schloemer, V. J. Schmithorst, J. N. Lorenz, R. S. Dunn, C. V. Vorhees, M. Wills-Karp, J. L. Degen, R. J. Davis, N. Mizushima, P. Rakic, B. J. Dardzinski, S. K. Holland, F. R. Sharp and C. Y. Kuan, “Cerebral Ischemia-Hypoxia Induces Intravascular Coagulation and Autophagy,” American Journal of Pathology, Vol. 169, No. 2, 2006, pp. 566-583. doi:10.2353/ajpath.2006.051066

[33]   W. Balduini, S. Carloni and G. Buonocore, “Autophagy in Hypoxia-Ischemia Induced Brain Injury: Evidence and Speculations,” Autophagy, Vol. 5, No. 2, 2009, pp. 221- 223. doi:10.4161/auto.5.2.7363

[34]   S. Barnes, “Evolution of the Health Benefits of Soy Isoflavones,” Proceedings of the Society for Experimental Biology and Medicine, Vol. 217, No. 3, 1998, pp. 386-392.

[35]   D. Altavilla, A. Crisafulli, H. Marini, M. Esposito, R. D. Anna, F. Corrado, A. Bitto and F. Squadrito, “Cardiovascular Effects of the Phytoestrogen Genistein,” Cardiovascular and Hematological Agents in Medicinal Chemistry, Vol 2, No. 2, 2004, pp. 179-186. doi:10.2174/1568016043477297

[36]   H. J. Wu and W. H. Chan, “Genistein Protects Methylglioxal-Induced Oxidative DNA Damage and Cell Injury in Human Mononuclear Cells,” Toxicology in Vitro, Vol. 21, No. 3, 2007, pp. 335-342. doi:10.1016/j.tiv.2006.09.002

[37]   M. S. Kindy, “Inhibition of Tyrosine Phosphorylation Prevents Delayed Neuronal Death Following Cerebral Ischemia,” Journal of Cerebral Blood Flow & Metabolism, Vol. 13, No.3, 1993, pp. 372-377.

[38]   R. Traystman, J. R. Kirsch and C. Koehler, “Oxygen Radicals Mechanism of Brain Injuria Following Ischemia and Reperfusion,” Journal of Applied Physiology, Vol. 71, No. 4, 1991, pp. 1185-1195.

[39]   Y. Matsuo, T. Kihara, M. Ikeda, M. Ninomiya, H. Onodera and K. Kogure, “Role of Platelet-Activating Factor and Thromboxane A2 in Radical Production during Ischemia and Reperfusion of the Rat Brain,” Brain Research, Vol. 709, No. 2, 1996, pp. 296-302. doi:10.1016/0006-8993(95)01324-5

[40]   T. Kimova and N. S. Chandel, “Mitochondrial Complex Iii Regulates Hypoxic Activation of HIF,” Cell Death and Differentiation, Vol. 15, No. 4, 2008, pp. 660-666. doi:10.1038/sj.cdd.4402307

[41]   P. H. Chan, “Role of Oxidants in Ischemic Brain Damage,” Stroke, Vol. 27, No. 6, 1996, pp. 1124-1129.

[42]   H. Friberg, T. Wieloch and R. F. Castilho, “Mitochondrial Oxidative Stress after Global Brain Ischemia in Rats,” Neuroscience Letters, Vol. 334, No. 2, 2002, pp. 111-114. doi:10.1016/S0304-3940(02)01116-3

[43]   M. A. Moro, A. Almeida, J. P. Bola?os and I. Lizasoain, “Mitochondrial Respiratory Chain and Free Radical Generation in Stroke,” Free Radical Biology & Medicine, Vol. 39, No. 10, 2005, pp. 1291-1304. doi:10.1016/j.freeradbiomed.2005.07.010

[44]   D. L. Zhang, Y. T. Zhang, J. J. Yin and B. L. Zhao, “Oral Administration of Crataegus Flavonoids Protects against Ischemia/Reperfusion Brain Damage in Gerbils,” Journal of Neurochemistry, Vol. 90, No. 1, 2004, pp. 211-219. doi:10.1111/j.1471-4159.2004.02480.x

[45]   V. E. Dosenko, V. S. Nagibin, L. V. Tumanovska and A. A. Moibenko, “Protective Effect of Autophagy in Anoxia-Reoxigenation of Isolated Cardiomyocyte?” Autophagy, Vol. 2, No. 4, 2006, pp. 305-306.

[46]   M. Komatsu, S. Waguri, T. Chiba, S. Murata, J. Iwata, I. Tanida, T. Ueno, M. Koike, Y. Uchiyama, E. Komonani and K. Tanaka, “Loss of Autophagy in the Central Nervous System Causes Neurodegeneration in Mice,” Nature, Vol. 441, No. 7095, 2006, pp. 880-884. doi:10.1038/nature04723

[47]   T. Hara, K. Nakamura, M. Matsui, A. Yamamoto, Y. Nakahara, R. Suzuki-Migishima, M. Yokoyama, K. Mishima, I. Saito, H. Okano and N. Mizushima, “Supression of Basal Autophagy in Neuronal Cells Causes Neurodegenerative Disease in Mice,” Nature, Vol. 441, No. 7095, 2006, pp. 885-889. doi:10.1038/nature04724

 
 
Top