Therapeutic effect of erythropoietin and its derivatives in the treatment of acute ischemic stroke

Abstract

Stroke is the third leading cause of death and disability in industrialized countries. The estimated costs of stroke to the healthcare system are $85 billion in the United States and $40 billion in the European Union. Despite the extensive research over the past decades, only therapies aimed at restoring blood flow to the affected area have been successful. However, the high risk of causing intracranial hemorrhage limits the application of this type of therapy to a small number of patients. Several studies have shown that, in addition to its well-known regulatory function in erythropoiesis, erythropoietin (EPO) is a potent neuroprotective agent against ischemic stroke. However, the use of EPO to treat stroke requires long-term protocols, high doses, and multiple administrations, which may cause thromboembolic complications due to increased hematocrit and blood viscosity, making EPO treatment unsuitable. To mitigate these adverse effects, various EPO analogues with neuroprotective properties but lacking erythropoietic activity have been investigated. This review aims to provide an overview of the protective mechanisms of EPO and its derivatives in the treatment of stroke.

Keywords

Acute ischemic stroke / excitotoxicity / oxidative stress / erythropoietin analogues / neuroprotection

References

[1] GBD 2019 Stroke Collaborators. Global, regional, and national burden of stroke and its risk factors, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet Neurol. 2021; 20: 795-820.

[2] Poomalai G, Prabhakar S, Jagadesh NS. Functional Ability and Health Problems of Stroke Survivors: An Explorative Study. Cureus. 2023; 15: e33375.

[3] Lindsay MP, Norrving B, Sacco RL, Brainin M, Hacke W, Martins S, et al. World Stroke Organization (WSO): Global Stroke Fact Sheet 2019. Int J Stroke. 2019; 14: 806-17.

[4] Brouns R, De Deyn PP. The complexity of neurobiological processes in acute ischemic stroke. Clin Neurol Neurosurg. 2009; 111: 483-95.

[5] Campbell BCV, Khatri P. Stroke. Lancet. 2020; 396: 129-42.

[6] Saini V, Guada L, Yavagal DR. Global Epidemiology of Stroke and Access to Acute Ischemic Stroke Interventions. Neurology. 2021; 97: S6-16.

[7] Jickling GC, Liu D, Stamova B, Ander BP, Zhan X, Lu A, et al. Hemorrhagic transformation after ischemic stroke in animals and humans. J Cereb Blood Flow Metab. 2014; 34: 185-99.

[8] Bernhardt J, Hayward KS, Kwakkel G, Ward NS, Wolf SL, Borschmann K, et al. Agreed definitions and a shared vision for new standards in stroke recovery research: The Stroke Recovery and Rehabilitation Roundtable taskforce. Int J Stroke. 2017; 12: 444-50.

[9] Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 1999; 22: 391-7.

[10] Lo EH, Dalkara T, Moskovitz MA. Mechanisms, challenges and opportunities in stroke. Nat Rev Neurosci. 2003; 4: 399-415.

[11] Moskowitz MA, Lo EH, Iadecola C. The science of stroke: mechanisms in search of treatments. Neuron. 2010; 67: 181-98.

[12] Erecińska M, Silver IA. Tissue oxygen tension and brain sensitivity to hypoxia. Respir Physiol. 2001; 128: 263-76.

[13] Astrup J, Siesjö BK, Symon L. Thresholds in Cerebral Ischemia - The Ischemic Penumbra. Stroke. 1981; 12: 723-5.

[14] Kidwell CS, Alger JR, Saver JL. Evolving paradigms in imaging of the ischemic penumbra with multimodal magnetic resonance imaging. Stroke. 2003; 34: 2729-35.

[15] Ermine CM, Bivard A, Parsons MW, Baron JC. The ischemic penumbra: From concept to reality. Int J Stroke. 2021; 16: 497-509.

[16] Hossmann KA. The two pathophysiologies of focal brain ischemia: implications for translational stroke research. J Cereb Blood Flow Metab. 2012; 32: 1310-6.

[17] Ramos-Cabrer P, Campos F, Sobrino T, Castillo J. Targeting the Ischemic Penumbra. Stroke. 2011; 42: S7-11.

[18] Lo EH. A new penumbra: transitioning from injury into repair after stroke. Nat Med. 2008; 14: 497-500.

[19] Ferrer I, Planas AM. Signaling of cell death and cell survival following focal cerebral ischemia: life and death struggle in the penumbra. J Neuropathol Exp Neurol. 2003; 62: 329-39.

[20] Ebinger M, De Silva DA, Christensen S, Parsons MW, Markus R, Donnan GA, et al. Imaging the penumbra - strategies to detect tissue at risk after ischemic stroke. J Clin Neurosci. 2009; 16: 178-87.

[21] El-Koussy M, Schroth G, Brekenfeld C, Arnold M. Imaging of acute ischemic stroke. Eur Neurol. 2014; 72: 309-16.

[22] Balch MHH, Nimjee SM, Rink C, Hannawi Y. Beyond the Brain: The Systemic Pathophysiological Response to Acute Ischemic Stroke. J Stroke. 2020; 22: 159-72.

[23] Zhao Y, Zhang X, Chen X, Wei Y. Neuronal injuries in cerebral infarction and ischemic stroke: From mechanisms to treatment (Review). Int J Mol Med. 2022; 49: 15.

[24] Singh TP, Weinstein JR, Murphy SP. Stroke: Basic and Clinical. Adv Neurobiol. 2017; 15: 281-93.

[25] Kuriakose D, Xiao Z. Pathophysiology and Treatment of Stroke: Present Status and Future Perspectives. Int J Mol Sci. 2020; 21: 7609.

[26] Feske SK. Ischemic Stroke. Am J Med. 2021; 134: 1457-64.

[27] Zauner A, Daugherty WP, Bullock MR, Wagner DS. Brain Oxygenation and Energy Metabolism. Neurosurgery. 2002; 51: 289-301.

[28] Attwell D, Laughlin SB. An Energy Budget for Signaling in the Grey Matter of the Brain. J Cereb Blood Flow Metab. 2001; 21: 1133-45.

[29] Bélanger M, Allaman I, Magistretti PJ. Brain Energy Metabolism: Focus on Astrocyte-Neuron Metabolic Cooperation. Cell Metab. 2011; 14: 724-38.

[30] Katsura K, Kristián T, Siesjö BK. Energy metabolism, ion homeostasis, and cell damage in the brain. Biochem Soc Trans. 1994; 22: 991-6.

[31] Martin RL, Lloyd HGE, Cowan AI. The early events of oxygen and glucose deprivation: setting the scene for neuronal death? Trends Neurosci. 1994; 17: 251-7.

[32] Rothman SM, Olney JW. Glutamate and the pathophysiology of hypoxic-ischemic brain damage. Ann Neurol. 1986; 19: 105-11.

[33] Belousov AB. Novel model for the mechanisms of glutamate-dependent excitotoxicity: role of neuronal gap junctions. Brain Res. 2012; 1487: 123-30.

[34] Kostandy BB. The role of glutamate in neuronal ischemic injury: the role of spark in fire. Neurol Sci. 2012; 33: 223-37.

[35] Rossi DJ, Oshima T, Attwell D. Glutamate release in severe brain ischaemia is mainly by reversed uptake. Nature. 2000; 403: 316-21.

[36] Camacho A, Massieu L. Role of Glutamate Transporters in the Clearance and Release of Glutamate during Ischemia and its Relation to Neuronal Death. Arch Med Res. 2006; 37: 11-8.

[37] Rama R, García JL. Excitotoxicity and Oxidative Stress in Acute Ischemic Stroke. In: Rodriguez J, Cesar J, editors. Acute Ischemic Stroke. Rijeka: IntechOpen; 2012.

[38] Olney JW. Brain Lesions, Obesity, and Other Disturbances in Mice Treated with Monosodium Glutamate. Science. 1969; 164: 719-21.

[39] Choi DW. Excitotoxic cell death. J Neurobiol. 1992; 23: 1261-76.

[40] Lai TW, Zhang S, Wang YT. Excitotoxicity and stroke: identifying novel targets for neuroprotection. Prog Neurobiol. 2014; 115: 157-88.

[41] Choi DW. Excitotoxicity: Still Hammering the Ischemic Brain in 2020. Front Neurosci. 2020; 14: 579953.

[42] Kritis AA, Stamoula EG, Paniskaki KA, Vavilis TD. Researching glutamate - induced cytotoxicity in different cell lines: a comparative/collective analysis/study. Front Cell Neurosci. 2015; 9: 91.

[43] Dong XX, Wang Y, Qin ZH. Molecular mechanisms of excitotoxicity and their relevance to pathogenesis of neurodegenerative diseases. Acta Pharmacol Sin. 2009; 30: 379-87.

[44] Prentice H, Modi JP, Wu JY. Mechanisms of Neuronal Protection against Excitotoxicity, Endoplasmic Reticulum Stress, and Mitochondrial Dysfunction in Stroke and Neurodegenerative Diseases. Oxid Med Cell Longev. 2015; 2015: 964518.

[45] Shen Z, Xiang M, Chen C, Ding F, Wang Y, Shang C, et al. Glutamate excitotoxicity: Potential therapeutic target for ischemic stroke. Biomed Pharmacother. 2022; 151: 113125.

[46] Zhou Y, Danbolt NC. Glutamate as a neurotransmitter in the healthy brain. J Neural Transm (Vienna). 2014; 121: 799-817.

[47] Willard SS, Koochekpour S. Glutamate, glutamate receptors, and downstream signaling pathways. Int J Biol Sci. 2013; 9: 948-59.

[48] Nedergaard M, Takano T, Hansen AJ. Beyond the role of glutamate as a neurotransmitter. Nat Rev Neurosci. 2002; 3: 748-55.

[49] Tani H, Dulla CG, Farzampour Z, Taylor-Weiner A, Huguenard JR, Reimer RJ. A local glutamate-glutamine cycle sustains synaptic excitatory transmitter release. Neuron. 2014; 81: 888-900.

[50] Mahmoud S, Gharagozloo M, Simard C, Gris D. Astrocytes Maintain Glutamate Homeostasis in the CNS by Controlling the Balance between Glutamate Uptake and Release. Cells. 2019; 8: 184.

[51] Norenberg MD. The Distribution of Glutamine Synthetase in the Rat Central Nervous System. J Histochem Cytochem. 1979; 27: 756-62.

[52] Nakanishi S. Molecular Diversity of Glutamate Receptors and Implications for Brain Function. Science. 1992; 258: 597-603.

[53] Lau A, Tymianski M. Glutamate receptors, neurotoxicity and neurodegeneration. Pflugers Arch. 2010; 460: 525-42.

[54] Hansen KB, Wollmuth LP, Bowie D, Furukawa H, Menniti FS, Sobolevsky AI, et al. Structure, Function, and Pharmacology of Glutamate Receptor Ion Channels. Pharmacol Rev. 2021; 73: 298-487.

[55] Hardingham GE, Fukunaga Y, Bading H. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat Neurosci. 2002; 5: 405-14.

[56] Brassai A, Suvanjeiev RG, Bán EG, Lakatos M. Role of synaptic and nonsynaptic glutamate receptors in ischaemia induced neurotoxicity. Brain Res Bull. 2015; 112: 1-6.

[57] Waxman EA, Lynch DR. N-methyl-D-aspartate Receptor Subtypes: Multiple Roles in Excitotoxicity and Neurological Disease. Neuroscientist. 2005; 11: 37-49.

[58] Ge Y, Chen W, Axerio-Cilies P, Wang YT. NMDARs in Cell Survival and Death: Implications in Stroke Pathogenesis and Treatment. Trends Mol Med. 2020; 26: 533-51.

[59] Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, et al. Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev. 2010; 62: 405-96.

[60] Paoletti P, Bellone C, Zhou Q. NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nat Rev Neurosci. 2013; 14: 383-400.

[61] Lynch DR, Guttmann RP. NMDA Receptor Pharmacology: Perspectives from Molecular Biology. Curr Drug Targets. 2001; 2: 215-31.

[62] Furukawa H, Singh SK, Mancusso R, Gouaux E. Subunit arrangement and function in NMDA receptors. Nature. 2005; 438: 185-92.

[63] Wu QJ, Tymianski M. Targeting NMDA receptors in stroke: new hope in neuroprotection. Mol Brain. 2018; 11: 15.

[64] Liu Y, Wong TP, Aarts M, Rooyakkers A, Liu L, Lai TW, et al. NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo. J Neurosci. 2007; 27: 2846-57.

[65] Sattler R, Xiong Z, Lu WY, Hafner M, Mac Donald JF, Tymianski M. Specific Coupling of NMDA Receptor Activation to Nitric Oxide Neurotoxicity by PSD-95 Protein. Science. 1999; 284: 1845-8.

[66] Kim E, Sheng M. PDZ domain proteins of synapses. Nat Rev Neurosci. 2004; 5: 771-81.

[67] Kornau HC, Schenker LT, Kennedy MB, Seeburg PH. Domain Interaction Between NMDA Receptor Subunits and the Postsynaptic Density Protein PSD-95. Science. 1995; 269: 1737-40.

[68] Chen X, Levy JM, Hou A, Winters C, Azzam R, Sousa AA, et al. PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density. Proc Natl Acad Sci U S A. 2015; 112: E6983-92.

[69] Niethammer M, Kim E, Sheng M. Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases. J Neurosci. 1996; 16: 2157-63.

[70] Brenman JE, Chao DS, Gee SH, McGee AW, Craven SE, Santillano DR, et al. Interaction of Nitric Oxide Synthase with the Postsynaptic Density Protein PSD-95 and α1-Syntrophin Mediated by PDZ Domains. Cell. 1996; 84: 757-67.

[71] Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury. Trends Neurosci. 1997; 20: 132-9.

[72] Bolaños JP, Almeida A. Roles of nitric oxide in brain hypoxia-ischemia. Biochim Biophys Acta. 1999; 1411: 415-36.

[73] Choi DW. Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage. Trends Neurosci. 1988; 11: 465-7.

[74] Brown GC. Nitric oxide and neuronal death. Nitric Oxide. 2010; 23: 153-65.

[75] Arundine M, Tymianski M. Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxicity. Cell Calcium. 2003; 34: 325-37.

[76] Aarts M, Liu Y, Liu L, Besshoh S, Arundine M, Gurd JW, et al. Treatment of Ischemic Brain Damage by Perturbing NMDA Receptor-PSD-95 Protein Interactions. Science. 2002; 298: 846-50.

[77] Sun HS, Doucette TA, Liu Y, Fang Y, Teves L, Aarts M, et al. Effectiveness of PSD95 Inhibitors in Permanent and Transient Focal Ischemia in the Rat. Stroke. 2008; 39: 2544-53.

[78] Szydlowska K, Tymiansky M. Calcium, ischemia and excitotoxicity. Cell Calcium. 2010; 47: 122-9.

[79] Siesjö BK, Zhao Q, Pahlmark K, Siesjö P, Katsura K, Folbergrová J. Glutamate, Calcium and Free Radicals as Mediators of Ischemic Brain Damage. Ann Thorac Surg. 1995; 59: 1316-20.

[80] Brini M, Calì T, Ottolini D, Carafoli E. Neuronal calcium signaling: function and dysfunction. Cell Mol Life Sci. 2014; 71: 2787-814.

[81] Mattson MP. Calcium and neurodegeneration. Aging Cell. 2007; 6: 337-50.

[82] Zündorf G, Reiser G. Calcium dysregulation and homeostasis of neural calcium in the molecular mechanisms of neurodegenerative diseases provide multiple targets for neuroprotection. Antioxid Redox Signal. 2011; 14: 1275-88.

[83] Tymianski M, Tator CH. Normal and Abnormal Calcium Homeostasis in Neurons: A Basis for the Pathophysiology of Traumatic and Ischemic Central Nervous System Injury. Neurosurgery. 1996; 38: 1176-95.

[84] Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calcium apoptosis link. Nat Rev Mol Cell Biol. 2003; 4: 552-65.

[85] Sattler R, Tymiansky M. Molecular mechanism of calcium-dependent excitotoxicity. J Mol Med. 2000; 78: 3-13.

[86] Bano D, Ankarcrona M. Beyond the critical point: An overview of excitotoxicity, calcium overload and the downstream consequences. Neurosci Lett. 2018; 663: 79-85.

[87] Venkatachalam K, Montell C. TRP channels. Annu Rev Biochem. 2007; 76: 387-417.

[88] Tymianski M. Emerging mechanisms of disrupted cellular signaling in brain ischemia. Nat Neurosci. 2011; 14: 1369-73.

[89] Xiong ZG, Zhu XM, Chu XP, Minami M, Hey J, Wei WL, et al. Neuroprotection in Ischemia: Blocking Calcium-Permeable Acid-Sensing Ion Channels. Cell. 2004; 118: 687-98.

[90] Knott AB, Bossy-Wetzel E. Nitric oxide in health and disease of the nervous system. Antioxid Redox Signal. 2009; 11: 541-54.

[91] Liu J, Liu MC, Wang KK. Calpain in the CNS: from synaptic function to neurotoxicity. Sci Signal. 2008; 1: re1.

[92] Starkov AA, Chinopoulos C, Fiskum G. Mitochondrial calcium and oxidative stress as mediators of ischemic brain injury. Cell Calcium. 2004; 36: 257-64.

[93] Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature. 2006; 443: 787-95.

[94] Duchen MR. Mitochondria, calcium-dependent neuronal death and neurodegenerative disease. Pflugers Arch. 2012; 464: 111-21.

[95] Niizuma K, Yoshioka H, Chen H, Kim GS, Jung JE, Katsu M, et al. Mitochondrial and apoptotic neuronal death signaling pathways in cerebral ischemia. Biochim Biophys Acta. 2010; 1802: 92-9.

[96] Sies H. Oxidative stress: a concept in redox biology and medicine. Redox Biol. 2015; 4: 180-3.

[97] Sies H, Belousov VV, Chandel NS, Davies MJ, Jones DP, Mann GE, et al. Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology. Nat Rev Mol Cell Biol. 2022; 23: 499-515.

[98] Tretter V, Hochreiter B, Zach ML, Krenn K, Klein KU. Understanding Cellular Redox Homeostasis: A Challenge for Precision Medicine. Int J Mol Sci. 2021; 23: 106.

[99] Pamplona R, Costantini D. Molecular and structural antioxidant defenses against oxidative stress in animals. Am J Physiol Regul Integr Comp Physiol. 2011; 301: R843-63.

[100] Valko M, Leibfritz D, Moncol J, Cronin MTD, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007; 39: 44-84.

[101] Sakamoto A, Ohnishi ST, Ohnishi T, Ogawa R. Relationship between free radical production and lipid peroxidation during ischemia-reperfusion injury in the rat brain. Brain Res. 1991; 554: 186-92.

[102] Berlett BS, Stadtman ER. Protein Oxidation in Aging, Disease, and Oxidative Stress. J Biol Chem. 1997; 272: 20313-6.

[103] Cui J, Holmes EH, Greene TG, Liu PK. Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain. FASEB J. 2000; 14: 955-67.

[104] Patel M. Targeting Oxidative Stress in Central Nervous System Disorders. Trends Pharmacol Sci. 2016; 37: 768-78.

[105] Kinuta Y, Kikuchi H, Ishikawa M, Kimura M, Itokawa Y. Lipid peroxidation in focal cerebral ischemia. J Neurosurg. 1989; 71: 421-9.

[106] Bedard K, Krause KH. The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology. Physiol Rev. 2007; 87: 245-313.

[107] Kusaka I, Kusaka G, Zhou C, Ishikawa M, Nanda A, Granger DN, et al. Role of AT₁ receptors and NAD(P)H oxidase in diabetes-aggravated ischemic brain injury. Am J Physiol Heart Circ Physiol. 2004; 286: H2442-51.

[108] Walder CE, Green SP, Darbonne WC, Mathias J, Rae J, Dinauer MC, et al. Ischemic Stroke Injury Is Reduced in Mice Lacking a Functional NADPH Oxidase. Stroke. 1997; 28: 2252-8.

[109] Chen H, Yoshioka H, Kim GS, Jung JE, Okami N, Sakata H, et al. Oxidative stress in ischemic brain damage: mechanisms of cell death and potential molecular targets for neuroprotection. Antioxid Redox Signal. 2011; 14: 1505-17.

[110] Castillo J, Rama R, Dávalos A. Nitric Oxide-Related Brain Damage in Acute Ischemic Stroke. Stroke. 2000; 31: 852-7.

[111] Fridovich I. Superoxide radical and superoxide dismutases. Annu Rev Biochem. 1995; 64: 97-112.

[112] Wang Y, Branicky R, Noë A, Hekimi S. Superoxide dismutases: Dual roles in controlling ROS damage and regulating ROS signaling. J Cell Biol. 2018; 217: 1915-28.

[113] Bell KF, Hardingham GE. CNS Peroxiredoxins and Their Regulation in Health and Disease. Antioxid Redox Signal. 2011; 14: 1467-77.

[114] Hanschmann E, Godoy JR, Berndt C, Hudemann C, Lillig CH. Thioredoxins, glutaredoxins, and peroxiredoxins-molecular mechanisms and health significance: from cofactors to antioxidants to redox signaling. Antioxid Redox Signal. 2013; 19: 1539-605.

[115] Lu J, Holmgren A. The thioredoxin antioxidant system. Free Radic Biol Med. 2014; 66: 75-87.

[116] Andreyev AY, Kushnareva YE, Starkov AA. Mitochondrial Metabolism of Reactive Oxygen Species. Biochemistry (Moscow). 2005; 70: 200-14.

[117] Angelova PR, Vinogradova D, Neganova ME, Serkova TP, Sokolov VV, Bachurin SO, et al. Pharmacological Sequestration of Mitochondrial Calcium Uptake Protects Neurons Against Glutamate Excitotoxicity. Mol Neurobiol. 2019; 56: 2244-55.

[118] Orellana-Urzúa S, Rojas I, Líbano L, Rodrigo R. Pathophysiology of Ischemic Stroke: Role of Oxidative Stress. Curr Pharm Des. 2020; 26: 4246-60.

[119] Moro MA, Cárdenas A, Hurtado O, Leza JC, Lizasoain I. Role of nitric oxide after brain ischaemia. Cell Calcium. 2004; 36: 265-75.

[120] Lundberg JO, Weitzberg E. Nitric oxide signaling in health and disease. Cell. 2022; 185: 2853-78.

[121] Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A. 1990; 87: 1620-4.

[122] Niizuma K, Endo H, Chan PH. Oxidative stress and mitochondrial dysfunction as determinants of ischemic neuronal death and survival. J Neurochem. 2009; 109: 133-8.

[123] Duan J, Gao S, Tu S, Lenahan C, Shao A, Sheng J. Pathophysiology and Therapeutic Potential of NADPH Oxidases in Ischemic Stroke-Induced Oxidative Stress. Oxid Med Cell Longev. 2021; 2021: 6631805.

[124] Chan PH. Reactive Oxygen Radicals in Signaling and Damage in the Ischemic Brain. J Cereb Blood Flow Metab. 2001; 21: 2-14.

[125] Allen CL, Bayraktutan U. Oxidative stress and its role in the pathogenesis of ischaemic stroke. Int J Stroke. 2009; 4: 461-70.

[126] Rodrigo R, Fernández-Gajardo R, Gutiérrez R, Matamala JM, Carrasco R, Miranda-Merchak A, et al. Oxidative Stress and Pathophysiology of Ischemic Stroke: Novel Therapeutic Opportunities. CNS Neurol Dis Drug Targets. 2013; 12: 698-714.

[127] Li G, Ye C, Zhu Y, Zhang T, Gu J, Pan J, et al. Oxidative Injury in Ischemic Stroke: A Focus on NADPH Oxidase 4. Oxid Med Cell Longev. 2022; 2022: 1148874.

[128] Culmsee C, Zhu C, Landshamer S, Becattini B, Wagner E, Pellecchia M, et al. Apoptosis-inducing factor triggered by poly(ADP-ribose) polymerase and Bid mediates neuronal cell death after oxygen-glucose deprivation and focal cerebral ischemia. J Neurosci. 2005; 25: 10262-72.

[129] Bakthavachalam P, Shanmugam PST. Mitochondrial dysfunction - Silent killer in cerebral ischemia. J Neurol Sci. 2017; 375: 417-23.

[130] Kroemer G, Galluzzi L, Brenner C. Mitochondrial Membrane Permeabilization in Cell Death. Physiol Rev. 2007; 87: 99-163.

[131] Bernardi P, Gerle C, Halestrap AP, Jonas EA, Karch J, Mnatsakanyan N, et al. Identity, structure, and function of the mitochondrial permeability transition pore: controversies, consensus, recent advances, and future directions. Cell Death Differ. 2023; 30: 1869-85.

[132] Bano D, Nicotera P. Ca²⁺ Signals and Neuronal Death in Brain Ischemia. Stroke. 2007; 38: 674-6.

[133] Ray SK. Currently Evaluated Calpain and Caspase Inhibitors for Neuroprotection in Experimental Brain Ischemia. Curr Med Chem. 2006; 13: 3425-40.

[134] Chandel NS. Evolution of Mitochondria as Signaling Organelles. Cell Metab. 2015; 22: 204-6.

[135] Nicholls DG. Mitochondrial calcium function and dysfunction in the central nervous system. Biochim Biophys Acta. 2009; 1787: 1416-24.

[136] Stuchebrukhov AA. Redox-Driven Proton Pumps of the Respiratory Chain. Biophys J. 2018; 115: 830-40.

[137] Zhao R, Jiang S, Zhang L, Yu Z. Mitochondrial electron transport chain, ROS generation and uncoupling (Review). Int J Mol Med. 2019; 44: 3-15.

[138] Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J. 1999; 341: 233-49.

[139] Halestrap AP. Calcium, mitochondria and reperfusion injury: a pore way to die. Biochem Soc Trans. 2006; 34: 232-7.

[140] Adam-Vizi V, Starkov AA. Calcium and mitochondrial reactive oxygen species generation: how to read the facts. J Alzheimers Dis. 2010; 20 Suppl 2: S413-26.

[141] Bernardi P, Forte M. The mitochondrial permeability transition pore. Novartis Found Symp. 2007;discussion 164-9.

[142] Halestrap AP. What is the mitochondrial permeability transition pore? J Mol Cell Cardiol. 2009; 46: 821-31.

[143] Abramov AY, Duchen MR. Mechanisms underlying the loss of mitochondrial membrane potential in glutamate excitotoxicity. Biochim Biophys Acta. 2008; 1777: 953-64.

[144] Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972; 26: 239-57.

[145] Almeida A, Bolaños JP. A transient inhibition of mitochondrial ATP synthesis by nitric oxide synthase activation triggered apoptosis in primary cortical neurons. J Neurochem. 2001; 77: 676-90.

[146] Fricker M, Tolkovsky AM, Borutaite V, Coleman M, Brown GC. Neuronal Cell Death. Physiol Rev. 2018; 98: 813-80.

[147] Hu ML, Pan YR, Yong YY, Liu Y, Yu L, Qin DL, et al. Poly (ADP-ribose) polymerase 1 and neurodegenerative diseases: Past, present, and future. Ageing Res Rev. 2023; 91: 102078.

[148] Dong M, Hu Q, Shen P, Pan J, Wei Y, Liu Y, et al. Recombinant Tissue Plasminogen Activator Induces Neurological Side Effects Independent on Thrombolysis in Mechanical Animal Models of Focal Cerebral Infarction: A Systematic Review and Meta-Analysis. PLoS One. 2016; 11: e0158848.

[149] Lees KR, Bluhmki E, von Kummer R, Brott TG, Toni D, Grotta JC, et al. Time to treatment with intravenous alteplase and outcome in stroke: an updated pooled analysis of ECASS, ATLANTIS, NINDS, and EPITHET trials. Lancet. 2010; 375: 1695-703.

[150] González RG, Furie KL, Goldmacher GV, Smith WS, Kamalian S, Payabvash S, et al. Good outcome rate of 35% in IV-tPA-treated patients with computed tomography angiography confirmed severe anterior circulation occlusive stroke. Stroke. 2013; 44: 3109-13.

[151] Tsivgoulis G, Katsanos AH, Sandset EC, Turc G, Nguyen TN, Bivard A, et al. Thrombolysis for acute ischaemic stroke: current status and future perspectives. Lancet Neurol. 2023; 22: 418-29.

[152] Renú A, Millán M, Román LS, Blasco J, Martí-Fàbregas J, Terceño M, et al.; CHOICE Investigators. Effect of Intra-arterial Alteplase vs Placebo Following Successful Thrombectomy on Functional Outcomes in Patients With Large Vessel Occlusion Acute Ischemic Stroke: The CHOICE Randomized Clinical Trial. JAMA. 2022; 327: 826-35.

[153] McCarthy DJ, Diaz A, Sheinberg DL, Snelling B, Luther EM, Chen SH, et al. Long-Term Outcomes of Mechanical Thrombectomy for Stroke: A Meta-Analysis. Sci World J. 2019; 2019: 7403104.

[154] Campbell BCV, Donnan GA, Lees KR, Hacke W, Khatri P, Hill MD, et al. Endovascular stent thrombectomy: the new standard of care for large vessel ischaemic stroke. Lancet Neurol. 2015; 14: 846-54.

[155] Barthels D, Das H. Current advances in ischemic stroke research and therapies. Biochim Biophys Acta Mol Basis Dis. 2020; 1866: 165260.

[156] O’Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH, Howells DW. 1,026 experimental treatments in acute stroke. Ann Neurol. 2006; 59: 467-77.

[157] Tymianski M. Can Molecular and Cellular Neuroprotection Be Translated Into Therapies for Patients? Yes, But Not the Way We Tried It Before. Stroke. 2010; 41: S87-90.

[158] Sutherland BA, Minnerup J, Balami JS, Arba F, Buchan AM, Kleinschnitz C. Neuroprotection for ischaemic stroke: translation from the bench to the bedside. Int J Stroke. 2012; 7: 407-18.

[159] Jelkmann W. Erythropoietin after a century of research: younger than ever. Eur J Haematol. 2007; 78: 183-205.

[160] Fisher JW, Koury S, Ducey T, Mendel S. Erythropoietin production by interstitial cells of hypoxic monkey kidneys. British J Haematol. 1996; 95: 27-32.

[161] Goldwasser E. Erythropoietin and the differentiation of red blood cells. Fed Proc. 1975; 34: 2285-92.

[162] Koury MJ, Bondurant MC. Erythropoietin Retards DNA Breakdown and Prevents Programmed Death in Erythroid Progenitor Cells. Science. 1990; 248: 378-81.

[163] Miyake T, Kung CKH, Goldwasser E. Purification of human erythropoietin. J Biol Chem. 1977; 252: 5558-64.

[164] Jacobs K, Shoemaker C, Rudersdorf R, Neill SD, Kaufman RJ, Mufson A, et al. Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature. 1985; 313: 806-10.

[165] Lin FK, Suggs S, Lin CH, Browne JK, Smalling R, Egrie JC, et al. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci U S A. 1985; 82: 7580-4.

[166] Winearls CG, Pippard MJ, Downing MR, Oliver DO, Reid C, Cotes PM. Effect of human erythropoietin derived from recombinant-DNA on the anaemia of patients maintained by chronic-haemodialysis. Lancet. 1986; 2: 1175-8.

[167] Wolcott DL, Marsh JT, Rue AL, Carr C, Nissenson AR. Recombinant human erythropoietin treatment may improve quality of life and cognitive function in chronic hemodialysis patients. Am J Kidney Dis. 1989; 14: 478-85.

[168] Jelkmann W. Physiology and pharmacology of erythropoietin. Transfus Med Hemother. 2013; 40: 302-9.

[169] Masuda S, Okano M, Yamagishi K, Nagao M, Ueda M, Sasaki R. A novel site of erythropoietin production. Oxygen-dependent production in cultured rat astrocytes. J Biol Chem. 1994; 269: 19488-93.

[170] Morishita E, Masuda S, Nagao M, Yasuda Y, Sasaki R. Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience. 1997; 76: 105-16.

[171] Brines M, Cerami A. Emerging biological roles for erythropoietin in the nervous system. Nat Rev Neurosci. 2005; 6: 484-97.

[172] Arcasoy MO. The non-haematopoietic biological effects of erythropoietin. Br J Haematol. 2008; 141: 14-31.

[173] Noguchi CT, Wang L, Rogers HM, Teng R, Jia Y. Survival and proliferative roles of erythropoietin beyond the erythroid lineage. Expert Rev Mol Med. 2008; 10: e36.

[174] Chateauvieux S, Grigorakaki C, Morceau F, Dicato M, Diederich M. Erythropoietin, erythropoiesis and beyond. Biochem Pharmacol. 2011; 82: 1291-303.

[175] Rey F, Balsari A, Giallongo T, Ottolenghi S, Giulio AMD, Samaja M, et al. Erythropoietin as a Neuroprotective Molecule: An Overview of Its Therapeutic Potential in Neurodegenerative Diseases. ASN Neuro. 2019; 11: 1759091419871420.

[176] Sakanaka M, Wen TC, Matsuda S, Masuda S, Morishita E, Nagao M, et al. In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc Natl Acad Sci U S A. 1998; 95: 4635-40.

[177] Sirén AL, Fratelli M, Brines M, Goemans C, Casagrande S, Lewczuk P, et al. Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proc Natl Acad Sci U S A. 2001; 98: 4044-9.

[178] Bernaudin M, Marti HH, Roussel S, Divoux D, Nouvelot A, MacKenzie ET, et al. A potential role for erythropoietin in focal permanent cerebral ischemia in mice. J Cereb Blood Flow Metab. 1999; 19: 643-51.

[179] Nguyen AQ, Cherry BH, Scott GF, Ryou M, Mallet RT. Erythropoietin: powerful protection of ischemic and post-ischemic brain. Exp Biol Med (Maywood). 2014; 239: 1461-75.

[180] Brines ML, Ghezzi P, Keenan S, Agnello D, de Lanerolle NC, Cerami C, et al. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc Natl Acad Sci U S A. 2000; 97: 10526-31.

[181] Wang Y, Zhang ZG, Rhodes K, Renzi M, Zhang RL, Kapke A, et al. Post-ischemic treatment with erythropoietin or carbamylated erythropoietin reduces infarction and improves neurological outcome in a rat model of focal cerebral ischemia. Br J Pharmacol. 2007; 151: 1377-84.

[182] Minnerup J, Heidrich J, Rogalewski A, Schäbitz W, Wellmann J. The efficacy of erythropoietin and its analogues in animal stroke models: a meta-analysis. Stroke. 2009; 40: 3113-20.

[183] Byts N, Sirén A. Erythropoietin: a multimodal neuroprotective agent. Exp Transl Stroke Med. 2009; 1: 4.

[184] Ehrenreich H, Kästner A, Weissenborn K, Streeter J, Sperling S, Wang KK, et al. Circulating damage marker profiles support a neuroprotective effect of erythropoietin in ischemic stroke patients. Mol Med. 2011; 17: 1306-10.

[185] Yip H, Tsai T, Lin H, Chen S, Sun C, Leu S, et al. Effect of erythropoietin on level of circulating endothelial progenitor cells and outcome in patients after acute ischemic stroke. Crit Care. 2011; 15: R40.

[186] Tsai T, Lu C, Wallace CG, Chang W, Chen S, Huang C, et al. Erythropoietin improves long-term neurological outcome in acute ischemic stroke patients: a randomized, prospective, placebo-controlled clinical trial. Crit Care. 2015; 19: 49.

[187] Mao R, Zong N, Hu Y, Chen Y, Xu Y. Neuronal Death Mechanisms and Therapeutic Strategy in Ischemic Stroke. Neurosci Bull. 2022; 38: 1229-47.

[188] Lai PH, Everett R, Wang FF, Arakawa T, Goldwasser E. Structural characterization of human erythropoietin. J Biol Chem. 1986; 261: 3116-21.

[189] Sasaki H, Bothner B, Dell A, Fukuda M. Carbohydrate structure of erythropoietin expressed in Chinese-hamster ovary cells by a human erythropoietin cDNA. J Biol Chem. 1987; 262: 12059-76.

[190] Weikert S, Papac D, Briggs J, Cowfer D, Tom S, Gawlitzek M, et al. Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins. Nat Biotechnol. 1999; 17: 1116-21.

[191] Spivak JL, Hogans BB. The in vivo metabolism of recombinant human erythropoietin in the rat. Blood. 1989; 73: 90-9.

[192] Elliott S, Egrie J, Browne J, Lorenzini T, Busse L, Rogers N, et al. Control of rHuEPO biological activity: the role of carbohydrate. Exp Hematol. 2004; 32: 1146-55.

[193] Powell JS, Berkner KL, Lebo RV, Adamson JW. Human erythropoietin gene: high level expression in stably transfected mammalian cells and chromosome localization. Proc Natl Acad Sci U S A. 1986; 83: 6465-9.

[194] Stockmann C, Fandrey J. Hypoxia induced erythropoietin production: a paradigm for oxygen-regulated gene expression. Clin Exp Pharmacol Physiol. 2006; 33: 968-79.

[195] Tomc J, Debeljak N. Molecular Insights into the Oxygen-Sensing Pathway and Erythropoietin Expression Regulation in Erythropoiesis. Int J Mol Sci. 2021; 22: 7074.

[196] Wang GL, Semenza GL. General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc Natl Acad Sci U S A. 1993; 90: 4304-8.

[197] Semenza GL. Involvement of oxygen-sensing pathways in physiologic and pathologic erythropoiesis. Blood. 2009; 114: 2015-9.

[198] Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell. 2012; 148: 399-408.

[199] Kaelin WG Jr, Ratcliffe PJ. Oxygen sensing by metazoans: The central role of the HIF hydroxylase pathway. Mol Cell. 2008; 30: 393-402.

[200] Hernández CC, Burgos CF, Gajardo AH, Silva-Grecchi T, Gavilan J, Toledo JR, et al. Neuroprotective effects of erythropoietin on neurodegenerative and ischemic brain diseases: the role of erythropoietin receptor. Neural Regen Res. 2017; 12: 1381-9.

[201] Shing KSCT, Broughton SE, Nero TL, Gillinder K, Ilsley MD, Ramshaw H, et al. EPO does not promote interaction between the erythropoietin and beta-common receptors. Sci Rep. 2018; 8: 12457.

[202] Ostrowski D, Heinrich R. Alternative Erythropoietin Receptors in the Nervous System. J Clin Med. 2018; 7: 24.

[203] Gassmann M, Heinicke K, Soliz J, Ogunshola OO. Non-erythroid functions of erythropoietin. Adv Exp Med Biol. 2003; 543: 323-30.

[204] Rama R, Garzón F, Rodríguez-Cruz Y, Maurice T, García-Rodríguez J. Neuroprotective effect of Neuro-EPO in neurodegenerative diseases: “Alea jacta est”. Neural Regen Res. 2019; 14: 1519-21.

[205] Vittori DC, Chamorro ME, Hernández YV, Maltaneri RE, Nesse AB. Erythropoietin and derivatives: Potential beneficial effects on the brain. J Neurochem. 2021; 158: 1032-57.

[206] Ma Y, Zhou Z, Yang G, Ding J, Wang X. The Effect of Erythropoietin and Its Derivatives on Ischemic Stroke Therapy: A Comprehensive Review. Front Pharmacol. 2022; 13: 743926.

[207] Rabie T, Marti HH. Brain protection by Erythropoietin: A Manifold Task. Physiology. 2008; 23: 263-74.

[208] Dang S, Liu X, Fu P, Gong W, Yan F, Han P, et al. Neuroprotection by local intra-arterial infusion of erythropoietin after focal cerebral ischemia in rats. Neurol Res. 2011; 33: 520-8.

[209] Cruz YR, Támos YM, Cernuda AM, Martines NS, González-Quevedo A, Testé IS, et al. Treatment with nasal neuro-EPO improves the neurological, cognitive, and histological state in a gerbil model of focal ischemia. ScientificWorldJournal. 2010; 10: 2288-300.

[210] Yuen CM, Leu S, Lee FY, Yen CH, Lin YC, Han P, et al. Erythropoietin Markedly Attenuates Brain Infarct Size and Improves Neurological Function in the Rat. J Invest Med. 2010; 58: 893-904.

[211] Ratilal BO, Arroja MMC, Rocha JPF, Fernandes AMA, Barateiro AP, Brites DMTO, et al. Neuroprotective effects of erythropoietin pretreatment in a rodent model of transient middle cerebral artery occlusion. J Neurosurg. 2014; 121: 55-62.

[212] Wang R, Zhao H, Li J, Duan Y, Fan Z, Tao Z, et al. Erythropoietin attenuates axonal injury after middle cerebral artery occlusion in mice. Neurol Res. 2017; 39: 545-51.

[213] Jerndal M, Forsberg K, Sena ES, Macleod MR, O’Collins VE, Linden T, et al. A systematic review and meta-analysis of erythropoietin in experimental stroke. J Cereb Blood Flow Metab. 2010; 30: 961-8.

[214] Yang J, Mukda S, Chen S. Diverse roles of mitochondria in ischemic stroke. Redox Biol. 2018; 16: 263-75.

[215] Ehrenreich H, Hasselblatt M, Dembowski C, Cepek L, Lewczuk P, Stiefel M, et al. Erythropoietin therapy for acute stroke is both safe and beneficial. Mol Med. 2002; 8: 495-505.

[216] Kilic E, Kilic U, Soliz J, Bassetti C, Gassmann M, Hermann DM. Brain-derived erythropoietin protects from focal cerebral ischemia by dual activation of ERK-1/-2 and Akt pathways. FASB J. 2005; 19: 2026-45.

[217] Si W, Wang J, Li M, Qu H, Gu R, Liu R, et al. Erythropoietin protects neurons from apoptosis via activating PI3K/AKT and inhibiting Erk1/2 signaling pathway. 3 Biotech. 2019; 9: 131.

[218] Chong ZZ, Shang YC, Mu Y, Cui S, Yao Q, Maiese K. Targeting erythropoietin for chronic neurodegenerative diseases. Expert Opin Ther Targets. 2013; 17: 707-20.

[219] Pardridge WM. The blood-brain barrier: bottleneck in brain drug development. NeuroRx. 2005; 2: 3-14.

[220] Yoo S, Cho B, Lee D, Son G, Lee Y, Han HS, et al. The erythropoietin-derived peptide MK-X and erythropoietin have neuroprotective effects against ischemic brain damage. Cell Death Dis. 2017; 8: e3003.

[221] Banks WA, Jumbe NL, Farrell CL, Niehoff ML, Heatherington AC. Passage of erythropoietic agents across the blood-brain barrier: a comparison of human and murine erythropoietin and the analog darbepoetin alfa. Eur J Pharmacol. 2004; 505: 93-101.

[222] Genc S, Kuralay F, Genc K, Akhisaroglu M, Fadiloglu S, Yorukoglu K, et al. Erythropoietin exerts neuroprotection in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated C57/BL mice via increasing nitric oxide production. Neurosci Lett. 2001; 298: 139-41.

[223] Sargin D, Friedrichs H, El-Kordi A, Ehrenreich H. Erythropoietin as neuroprotective and neuroregenerative treatment strategy: Comprehensive overview of 12 years of preclinical and clinical research. Best Pract Res Clin Anaesthesiol. 2010; 24: 573-94.

[224] Zhang F, Xing J, Liou AK, Wang S, Gan Y, Luo Y, et al. Enhanced Delivery of Erythropoietin Across the Blood-Brain Barrier for Neuroprotection against Ischemic Neuronal Injury. Transl Stroke Res. 2010; 1: 113-21.

[225] Asadi B, Askari GR, Khorvash F, Bagherpur A, Mehrabi F, Karimi M, et al. Neuroprotective effects of erythropoietin in acute ischemic stroke. Int J Prev Med. 2013; 4: S306-12.

[226] Cantarelli C, Angeletti A, Cravedi P. Erythropoietin, a multifaceted protein with innate and adaptive immune modulatory activity. Am J Transplant. 2019; 19: 2407-14.

[227] Malerba F, Paolett F, Capsoni S, Cattaneo A. Intranasal delivery of therapeutic proteins for neurological diseases. Expert Opin Drug Deliv. 2011; 8: 1277-96.

[228] Garcia-Rodriguez JC, Sosa-Teste I. The nasal route as a potential pathway for delivery of erythropoietin in the treatment of acute ischemic stroke in humans. ScientificWorldJournal. 2009; 9: 970-81.

[229] Genc S, Zadeoglulari Z, Oner MG, Genc K, Digicaylioglu M. Intranasal erythropoietin therapy in nervous system disorders. Expert Opin Drug Deliv. 2011; 8: 19-32.

[230] Teste IS, Tamos YM, Cruz YR, Cernada AM, Rodríguez JC, Martínez NS, et al. Dose effect evaluation and therapeutic window of the neuro-EPO nasal application for the treatment of the focal ischemia model in the Mongolian gerbil. ScientificWorldJournal. 2012; 2012: 607498.

[231] Yu YP, Xu QQ, Zhang Q, Zhang WP, Zhang LH, Wei EQ. Intranasal recombinant human erythropoietin protects rats against focal cerebral ischemia. Neurosci Lett. 2005; 387: 5-10.

[232] Macias-Velez RJ, Fukushima-Díaz de León L, Beas-Zárate C, Rivera-Cervantes MC. Intranasal Erythropoietin Protects CA1 Hippocampal Cells, Modulated by Specific Time Pattern Molecular Changes After Ischemic Damage in Rats. J Mol Neurosci. 2019; 68: 590-602.

[233] Gross AW, Lodish HF. Cellular Trafficking and Degradation of Erythropoietin and Novel Erythropoiesis Stimulating Protein (NESP). J Biol Chem. 2006; 281: 2024-32.

[234] Ehrenreich H, Weissenborn K, Prange H, Schneider D, Weimar C, Wartenberg K, et al.; EPO Stroke Trial Group. Recombinant Human Erythropoietin in the Treatment of Acute Ischemic Stroke. Stroke. 2009; 40: e647-56.

[235] Krapf R, Hulter HN. Arterial Hypertension Induced by Erythropoietin and Erythropoiesis-Stimulating Agents (ESA). Clin J Am Soc Nephrol. 2009; 4: 470-80.

[236] Leist M, Ghezzi P, Grasso G, Bianchi R, Villa P, Fratelli M, et al. Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science. 2004; 305: 239-42.

[237] Erbayraktar S, Grasso G, Sfacteria A, Xie Q, Coleman T, Kreilgaard M, et al. Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo. Proc Natl Acad Sci U S A. 2003; 100: 6741-6.

[238] Lapchak PA. Carbamylated erythropoietin to treat neuronal injury: new development strategies. Expert Opin Investig Drugs. 2008; 17: 1175-86.

[239] Chen J, Yang Z, Zhang X. Carbamylated Erythropoietin: A Prospective Drug Candidate for Neuroprotection. Biochem Insights. 2016; 8: 25-9.

[240] Chamorro ME, Wenker SD, Vota DM, Vittori DC, Nesse AB. Signaling pathways of cell proliferation are involved in the differential effect of erythropoietin and its carbamylated derivative. Biochem Biophys Acta. 2013; 1833: 1960-8.

[241] Santos-Morales O, Díaz-Machado A, Jiménez-Rodríguez D, Pomares-Iturralde Y, Festary-Casanovas T, González-Delgado CA, et al. Nasal administration of the neuroprotective candidate NeuroEPO to healthy volunteers: a randomized, parallel, open-label safety study. BMC Neurol. 2017; 17: 129.

[242] Garzón F, Rodríguez Y, García JC, Rama R. Neuroprotective Effects of NeuroEPO Using an In Vitro Model of Stroke. Behav Sci (Basel). 2018; 8: 26.

[243] Garzón F, Coimbra D, Parcerisas A, Rodríguez Y, García JC, Soriano S, et al. NeuroEPO Preserves Neurons from Glutamate-Induced Excitotoxicity. J Alzheimers Dis. 2018; 65: 1469-83.

[244] Cheung NS, Pascoe CJ, Giardina SF, John CA, Beart PM. Micromolar L-glutamate induces extensive apoptosis in an apoptoptic-necrotic continuum of insult-dependent, excitotoxic injury in cultured cortical neurones. Neuropharmacol. 1998; 37: 1419-29.

[245] Zhang Y, Bhavnani BR. Glutamate-induced apoptosis in primary cortical neurons is inhibited by equine estrogens via down-regulation of caspase-3 and prevention of mitochondrial cytochrome c release. BMC Neurosci. 2005; 6: 13.

[246] Choi DW, Maulucci-Gedde M, Kriegstein AR. Glutamate neurotoxicity in cortical cell culture. J Neurosci. 1987; 7: 357-68.

[247] D’Orsi B, Mateyka J, Prehn JHM. Control of mitochondrial physiology and cell death by the Bcl-2 family proteins Bax and Bok. Neurochem Int. 2017; 109: 162-70.

[248] García-Llano M, Pedroso-Ibáñez I, Morales-Chacón L, Rodríguez-Obaya T, Pérez-Ruiz L, Sosa-Testé I, et al. Short-Term Tolerance of Nasally-Administered NeuroEPO in Patients with Parkinson Disease. MEDICC Rev. 2021; 23: 49-54.

[249] García-Artalejo JA, Mancera-Arteu M, Sanz-Nebot V, Rodríguez T, Giménez E. Characterizing a novel hyposialylated erythropoietin by intact glycoprotein and glycan analysis. J Pharm Biomed Anal. 2022; 213: 114686.