Mechanisms of Erythropoietin-induced Brain Protection in Neonatal Hypoxia-Ischemia Rat Model

Erythropoietin, a hemotopoietic growth factor, has brain protective actions. This study investigated the mechanisms of Recombinant Human EPO (rhEPO)-induced brain protection in neonates. An established rat hypoxia-ischemia model was used by ligation of the right common carotid artery of 7-day-old pups, followed by 90 minute of hypoxia (8% 02 and 92% N2) at 37°C. Animals were divided into three groups: control, hypoxia-ischemia, and hypoxia-ischemia plus rhEPO treatment. In rhEPO treated pups, 300 units rhEPO was administered intraperitoneally 24 hours before hypoxia. rhEPO treatment (300 units) was administered daily for an additional 2 days. ELISA and immunohistochemistry examined the expression of EPO and EPOR. Brain weight, morphology, TUNEL assay, and DNA laddering evaluated brain protection. rhEPO abolished mortality (from 19% to 0%) during hypoxia insult, increased brain weight from 52% to 88%, reduced DNA fragmentation, and decreased TUNEL-positive cells. Real-time RT-PCR, Western blot, and immunohistochemistry revealed an enhanced expression of heat shock protein 27 (HSP27) in ischemic brain hemisphere. Double labeling of TUNEL with HSP27 showed most HSP27 positive cells were negative to TUNEL staining. rhEPO reduces brain injury, especially apoptotic cell death after neonatal hypoxia-ischemia, partially mediated by the activation of HSP27.

[1]  Pierre J Magistretti,et al.  Lactate is a Preferential Oxidative Energy Substrate over Glucose for Neurons in Culture , 2003, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[2]  Ç. Ulukuş,et al.  Neuroprotective Effect of Erythropoietin on Hypoxic-Ischemic Brain Injury in Neonatal Rats , 2003, Neonatology.

[3]  M. Buemi,et al.  The Pleiotropic Effects of Erythropoietin in the Central Nervous System , 2003, Journal of neuropathology and experimental neurology.

[4]  F. Barone,et al.  Caspase 3 activation is essential for neuroprotection in preconditioning , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[5]  John H. Zhang,et al.  A possible role of RhoA/Rho-kinase in experimental spinal cord injury in rat , 2003, Brain Research.

[6]  J. Fisher,et al.  Erythropoietin: Physiology and Pharmacology Update 2 , 2003, Experimental biology and medicine.

[7]  Kenneth Maiese,et al.  Erythropoietin Is a Novel Vascular Protectant Through Activation of Akt1 and Mitochondrial Modulation of Cysteine Proteases , 2002, Circulation.

[8]  T. Eid,et al.  Recombinant human erythropoietin for neuroprotection: what is the evidence? , 2002, Clinical breast cancer.

[9]  U. Dirnagl,et al.  Erythropoietin Is a Paracrine Mediator of Ischemic Tolerance in the Brain: Evidence from an In Vitro Model , 2002, The Journal of Neuroscience.

[10]  J. LaManna,et al.  Activation of Hypoxia-Inducible Factor-1 in the Rat Cerebral Cortex after Transient Global Ischemia: Potential Role of Insulin-Like Growth Factor-1 , 2002, The Journal of Neuroscience.

[11]  P. Ghezzi,et al.  Erythropoietin exerts an anti-inflammatory effect on the CNS in a model of experimental autoimmune encephalomyelitis , 2002, Brain Research.

[12]  John H. Zhang,et al.  Hyperbaric oxygenation prevented brain injury induced by hypoxia–ischemia in a neonatal rat model , 2002, Brain Research.

[13]  M. Buemi,et al.  Erythropoietin and the brain: from neurodevelopment to neuroprotection. , 2002, Clinical science.

[14]  John H. Zhang,et al.  Mechanism of RhoA/Rho kinase activation in endothelin-1- induced contraction in rabbit basilar artery. , 2002, American journal of physiology. Heart and circulatory physiology.

[15]  S. Juul,et al.  Erythropoietin in the central nervous system, and its use to prevent hypoxic‐ischemic brain damage , 2002, Acta paediatrica (Oslo, Norway : 1992). Supplement.

[16]  P. Lewczuk,et al.  Erythropoietin Therapy for Acute Stroke Is Both Safe and Beneficial , 2002, Molecular medicine.

[17]  John H. Zhang,et al.  Oxyhemoglobin produces necrosis, not apoptosis, in astrocytes , 2002, Brain Research.

[18]  D. Butterfield,et al.  Molecular Chaperones and Their Roles in Neural Cell Differentiation , 2002, Developmental Neuroscience.

[19]  A. Cerami,et al.  Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[20]  C. Grimm,et al.  HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration , 2002, Nature Medicine.

[21]  K. Maiese,et al.  Hematopoietic Factor Erythropoietin Fosters Neuroprotection through Novel Signal Transduction Cascades , 2002, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[22]  E. Mackenzie,et al.  Normobaric Hypoxia Induces Tolerance to Focal Permanent Cerebral Ischemia in Association with an Increased Expression of Hypoxia-Inducible Factor-1 and its Target Genes, Erythropoietin and VEGF, in the Adult Mouse Brain , 2002, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[23]  J. McIntyre,et al.  Recent advances in neonatology , 2002, Current opinion in obstetrics & gynecology.

[24]  R. Hata,et al.  Erythropoietin protects neurons against chemical hypoxia and cerebral ischemic injury by up‐regulating Bcl‐xL expression , 2002, Journal of neuroscience research.

[25]  A. Ruello,et al.  Recombinant human erythropoietin (rHuEPO): more than just the correction of uremic anemia. , 2002, Journal of nephrology.

[26]  F. Tomasello,et al.  Does administration of recombinant human erythropoietin attenuate the increase of S-100 protein observed in cerebrospinal fluid after experimental subarachnoid hemorrhage? , 2002, Journal of neurosurgery.

[27]  E. Wintour,et al.  Quantitation of the mRNA levels of Epo and EpoR in various tissues in the ovine fetus , 2002, Molecular and Cellular Endocrinology.

[28]  F. Squadrito,et al.  Erythropoietin prevents cognition impairment induced by transient brain ischemia in gerbils. , 2002, European journal of pharmacology.

[29]  A. Cerami,et al.  Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[30]  S. Ashwal,et al.  Animal models of neonatal stroke , 2001, Current opinion in pediatrics.

[31]  D. Ferriero.  Oxidant Mechanisms in Neonatal Hypoxia-Ischemia , 2001, Developmental Neuroscience.

[32]  Stuart A. Lipton,et al.  Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-κB signalling cascades , 2001, Nature.

[33]  A. Cerami,et al.  Beyond erythropoiesis: novel applications for recombinant human erythropoietin. , 2001, Seminars in hematology.

[34]  S. Raz,et al.  Neonatal hypoxic risk in preterm birth infants: the influence of sex and severity of respiratory distress on cognitive recovery. , 2001, Neuropsychology.

[35]  John H. Zhang,et al.  Oxyhemoglobin induces caspase‐mediated cell death in cerebral endothelial cells , 2001, Journal of neurochemistry.

[36]  Changlian Zhu,et al.  Synergistic Activation of Caspase-3 by m-Calpain after Neonatal Hypoxia-Ischemia , 2001, The Journal of Biological Chemistry.

[37]  S. Juul,et al.  The Biology of Erythropoietin in the Central Nervous System and Its Neurotrophic and Neuroprotective Potential , 2001, Neonatology.

[38]  P. Lewczuk,et al.  Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[39]  M. Hori,et al.  Protective and regenerative response endogenously induced in the ischemic brain. , 2001, Canadian journal of physiology and pharmacology.

[40]  G. Grasso,et al.  Neuroprotective effect of recombinant human erythropoietin in experimental subarachnoid hemorrhage. , 2001, Journal of neurosurgical sciences.

[41]  John H. Zhang,et al.  Caspase Inhibitors Attenuate Oxyhemoglobin-Induced Apoptosis in Endothelial Cells , 2001, Stroke.

[42]  J. Fandrey,et al.  Erythropoietin gene expression in different areas of the developing human central nervous system. , 2000, Brain research. Developmental brain research.

[43]  V. Miller.  Neonatal cerebral infarction. , 2000, Seminars in pediatric neurology.

[44]  F. Tomasello,et al.  Effect of recombinant human erythropoietin on cerebral ischemia following experimental subarachnoid hemorrhage. , 2000, European journal of pharmacology.

[45]  M. Bernaudin,et al.  Neuroprotection and Angiogenesis: Dual Role of Erythropoietin in Brain Ischemia. , 2000, News in physiological sciences : an international journal of physiology produced jointly by the International Union of Physiological Sciences and the American Physiological Society.

[46]  H. Mohrenweiser,et al.  Production and processing of erythropoietin receptor transcripts in brain. , 2000, Brain research. Molecular brain research.

[47]  P Ghezzi,et al.  Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[48]  G. Semenza,et al.  Role of hypoxia‐inducible factor‐1 in hypoxia‐induced ischemic tolerance in neonatal rat brain , 2000, Annals of neurology.

[49]  S. Juul,et al.  Nonerythropoietic roles of erythropoietin in the fetus and neonate. , 2000, Clinics in perinatology.

[50]  M. Gassmann,et al.  Epolones induce erythropoietin expression via hypoxia-inducible factor-1α activation , 2000 .

[51]  A. Allegra,et al.  Erythropoietin protects against brain ischemic injury by inhibition of nitric oxide formation. , 2000, European journal of pharmacology.

[52]  A. Allegra,et al.  Intravenous recombinant erythropoietin does not lead to an increase in cerebrospinal fluid erythropoietin concentration. , 2000, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[53]  S. Juul,et al.  Erythropoietin in the Cerebrospinal Fluid of Neonates Who Sustained CNS Injury , 1999, Pediatric Research.

[54]  C. Wagner,et al.  The use of hypothermia: a role in the treatment of neonatal asphyxia? , 1999, Pediatric neurology.

[55]  J. Rennie,et al.  Recent advances in neonatology , 1999, Archives of disease in childhood. Fetal and neonatal edition.

[56]  E. Mackenzie,et al.  A Potential Role for Erythropoietin in Focal Permanent Cerebral Ischemia in Mice , 1999, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[57]  S. Juul,et al.  Erythropoietin in the neonate. , 1999, Current problems in pediatrics.

[58]  A. Rojiani,et al.  Immunohistochemical Localization of Erythropoietin and Its Receptor in the Developing Human Brain , 1999, Pediatric and developmental pathology : the official journal of the Society for Pediatric Pathology and the Paediatric Pathology Society.

[59]  J. Connor,et al.  Rat model of perinatal hypoxic‐ischemic brain damage , 1999, Journal of neuroscience research.

[60]  A. Shah,et al.  Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury. , 1998, The Journal of clinical investigation.

[61]  E. Morishita,et al.  In vivo evidence that erythropoietin protects neurons from ischemic damage. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[62]  R. Vannucci,et al.  A model of Perinatal Hypoxic‐Ischemic Brain Damage a , 1997, Annals of the New York Academy of Sciences.

[63]  S. Peterec,et al.  Management of neonatal Rh disease. , 1995, Clinics in perinatology.

[64]  M. Gassmann,et al.  Localization of specific erythropoietin binding sites in defined areas of the mouse brain. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[65]  R. Burke,et al.  Localization of c-fos, c-jun, and hsp70 mRNA expression in brain after neonatal hypoxia-ischemia. , 1994, Brain research. Developmental brain research.

[66]  T. Nowak,et al.  The Heat Shock/Stress Response in Focal Cerebral Ischemia , 1994, Brain pathology.

[67]  K. Kogure,et al.  Altered Gene Expression in Cerebral Ischemia , 1993, Stroke.

[68]  R. Burke,et al.  Immediate early gene induction after neonatal hypoxia-ischemia. , 1993, Brain research. Molecular brain research.

[69]  M. Goldberg,et al.  Hypoxia up-regulates the activity of a novel erythropoietin mRNA binding protein. , 1991, The Journal of biological chemistry.

[70]  R. Vannucci.  Current and potentially new management strategies for perinatal hypoxic-ischemic encephalopathy. , 1990, Pediatrics.

[71]  D. Ferriero,et al.  Hypoxia-ischemia induces heat shock protein-like (HSP72) immunoreactivity in neonatal rat brain. , 1990, Brain research. Developmental brain research.

[72]  B. Dwyer,et al.  Synthesis of the major inducible heat shock protein in rat hippocampus after neonatal hypoxia-ischemia , 1989, Experimental Neurology.

[73]  K. Raivio,et al.  Prediction of perinatal brain damage by cord plasma vasopressin, erythropoietin, and hypoxanthine values. , 1988, The Journal of pediatrics.

[74]  J. Rice,et al.  The influence of immaturity on hypoxic‐ischemic brain damage in the rat , 1981, Annals of neurology.

[75]  E. Kaptanoğlu,et al.  Erythropoietin prevents ischemia-reperfusion from inducing oxidative damage in fetal rat brain , 2002, Child's Nervous System.

[76]  G. Acs,et al.  Erythropoietin and erythropoietin receptor expression in human cancer. , 2001, Cancer research.

[77]  A. Mureşan,et al.  The brain and other sites of erythropoietin production. , 2000, Romanian journal of physiology : physiological sciences.

[78]  M. Gassmann,et al.  Epolones induce erythropoietin expression via hypoxia-inducible factor-1 alpha activation. , 2000, Blood.

[79]  John H. Zhang,et al.  Oxyhemoglobin produces necrosis in cultured smooth muscle cells. , 2000, Acta neurochirurgica. Supplement.

[80]  R. Keep,et al.  Thrombin preconditioning, heat shock proteins and thrombin-induced brain edema. , 2000, Acta neurochirurgica. Supplement.

[81]  貞本 泰孝.  Erythropoietin Prevents Place Navigation Disability and Cortical Infarction in Rats with Permanent Occlusion of the Middle Cerebral Artery , 2000 .

[82]  Yan Li,et al.  Erythropoietin and Erythropoietin Receptor in the Developing Human Central Nervous System , 1998, Pediatric Research.

[83]  K. Eckardt.  The ontogeny of the biological role and production of erythropoietin , 1995, Journal of perinatal medicine.

[84]  J. Goddard-finegold,et al.  Cerebrovascular regulation and neonatal brain injury. , 1991, Pediatric neurology.

[85]  A. Maxwell,et al.  Subcutaneous erythropoietin therapy and hypertensive encephalopathy. , 1990, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.