The term neuroprotection refers to mechanisms within the nervous system which protect neurons from apoptosis or degeneration, for example following a brain injury or as a result of chronic neurodegenerative diseases. The word derives from the words "neuron" (Greek for nerve cell) and "protection" (Latin for "saving").
Currently, there is a broad interest in how apoptosis and neuroprotection act on the brain in situations as different as growing up and learning or being ill (stroke, schizophrenia, Parkinson's disease).
A recent post-mortem study of the anterior cingulate cortex of persons with schizophrenia found increased levels of cellular signaling proteins, primarily PEBP, that may lead to increased levels of neuroprotection. (Clark, 2006)
Neuroprotection: Introduction to Erythropoietin and Erythrogenesis
Erythropoietin (Epo) is a glycoprotein that controls erythropoiesis, or red blood cell production. It is synthesized by renal peritubular cells in adults, with a small amount being produced in the liver (Jacobson 1957; Fisher 1996). It binds to the erythropoietin receptor (EpoR) on the red cell surface and activates a JAK2 cascade. This receptor is also found in a large number of tissues such as bone marrow cells, lymphocytes, and peripheral/central nerve cells, many of which activate intracellular biological pathways upon binding with Epo.
Primary Role in Red Cell Blood Line
Erythropoietin has its primary effect on red blood cells by promoting red blood cell survival through protecting these cells from apoptosis. It also cooperates with various growth factors involved in the development of precursor red cells. It has a range of actions including vasoconstriction-dependent hypertension, stimulating angiogenesis, and inducing proliferation of smooth muscle fibers.
History of Erythropoietin Research
Discovery of Erythropoietin
In 1906 Paul Carnot, a Professor of Medicine in Paris, and his assistant DeFlandre proposed the idea that erythropoiesis was regulated by hormones. After conducting experiments on rabbits subject to bloodletting, Carnot and DeFlandre attributed an increase in red blood cells in rabbit subjects to a hemotopic factor called hemopoietin. Eva Bonsdorff and Eeva Jalavisto continued to study red cell production and later called the hemopoietic substance ‘erythropoietin.’ Further studies investigating the existence of EPO by Reissman and Erslev demonstrated that a certain substance circulated in the blood was able to stimulate red blood cell production and increase hematocrit. This substance was finally purified and confirmed as erythropoietin, opening doors to therapeutic uses for Epo in diseases like anemia (Jelkmann 2007; Hoke 2006).
In 1985, Lin et al. isolated the human erythropoietin gene from a genomic phage library and were able to characterize it for research and production (Lin 1985). Their research demonstrated that the gene for erythropoietin encoded the production of Epo in mammalian cells that is biologically active in vitro and in vivo. This opened up the door for the industrial production of recombinant erythropoietin (RhEpo) for treating anemia patients.
Novel Erythropoiesis Stimulating Protein
More recently a novel erythropoiesis stimulating protein (NESP) has been produced (MacDougall 2000). This glycoprotein demonstrates anti-anemic capabilities and has a longer terminal half-life than erythropoietin. NESP offers chronic renal failure patients a lower dose of hormones to maintain normal hemoglobin levels.
Erythropoietin and the Central Nervous System
Erythropoietin and its receptor are both present in the central nervous system with erythropoietin alpha capable of crossing the blood brain barrier via active transport (Juul 1998). This was demonstrated by Juul et al. in their experiment to show the presence of Epo within the spinal fluid of infants and the presence of Epo-R in the spinal cord. Grimm et al reported on the detection of Epo and Epo-R expression in the mammalian retina, and a potential therapy to protect photoreceptors via hypoxic pretreatment (Grimm 2002).
The administration of erythropoietin protecting nerve cells from hypoxia-induced glutamate toxicity was demonstrated in experiments by Grimm et al in 2002 and Morishita et al in 1997 (Grimm 2002, Morishita 1997). Grimm and his collaborators showed that acute hypoxia inducement in the adult mouse retina stimulates expression of Epo in addition to other growth factors. Epo response is stimulated by hypoxia and is capable of protecting against apoptosis of erythroid progenitors via a mechanism that is described in the Mechanism of Action section (Grimm 2002). The work of Morishita and his colleagues has provided evidence for the presence of Epo-R in cultured hippocampal and cerebral cortical neurons in rat embryos. Epo was capable of protecting the cultured neurons from glutamate neurotoxicity after only a short exposure. They concluded that since erythropoietin exposure induces increases in intracellular calcium concentration, it must play a neuroprotective role in brain injury from hypoxia or ischemia (Morishita 1997).
Erythropoietin’s role in reducing immune response has been described recently by Michael Brines through his research in administering recombinant human Epo into the blood circulation, which was translocated to the cerebrospinal fluid. RhEpo administer in rats prevented apoptosis of neurons during a cerebral arterial occlusion. It also reduced infarction volume by 75% and decreased post-infarct inflammation (Brines 2002).
Epo has also been demonstrated to enhance nerve recovery after spinal trauma. Celik and associates investigated motor neuron apoptosis in rabbits with a transient global spinal ischemia model (Celik 2002). This allowed them to test if administered Epo can cross the blood-spinal cord barrier and protect motor neurons in the spinal cord. They produced spinal cord ischemia in rabbits and administered RhEpo. After comparison with a control administration of saline, the functional neurological status of animals given RhEpo was better after recovery from anesthesia, and kept improving over a two day period. The animals given saline demonstrated a poor functional neurological status and showed no significant improvements. These results showed that RhEpo has both an acute and delayed beneficial action in ischemic spinal cord injury.
Development with Mutant Epo and EpoR
While EpoR has been found in high levels in the embryonic brain, its role in brain development is unclear. Yu and colleagues proved evidence that Epo stimulates neural progenitor cells and prevents apoptosis in the embryonic brain using mice model experiments (Yu 2002). Mice without EpoR demonstrated severe anemia, defective heart development, and eventually death around embryonic day 13.5 from apoptosis in the liver, endocardium, myocardium, and fetal brain. As early as embryonic day 10.5 the lack of EpoR can affect brain development by increasing fetal brain apoptosis and decreasing the number of neural progenitor cells. By exposing cultures of EpoR positive embryonic cortical neurons to stimulation by Epo administration, the cells decreased apoptosis, as opposed to the decrease in neuron generation in EpoR negative cells. Yu et al demonstrated that the neuroprotective activity of Epo can be observed as early as embryonic day 10.5 in the developing brain and contributes to selective cell survival in the developing brain.
However, Suzuki and colleagues questioned whether EpoR may or may not be a determining factor for the nervous system function (Suzuki 2002). They suggest that the contribution of Epo and EpoR to neuroprotection and development are not as clearly understood as its role in erythropoiesis in hematopoietic tissue. This group studied a line of mice that expressed EpoR exclusively in hematopoietic cells and found that the mice developed normally and were fertile, despite the lack of EpoR in nonhematopoietic tissue. They analyzed this line of mice and found differential expression of EpoR between erythroid cells. Most notably, they found that plasma Epo concentration is regulated by nonhematopoietic EpoR expression when they timed the peak of plasma concentrations for induced anemia in mutant and wild-type mice. As such, they concluded that the expression of EpoR in nonhematopoietic tissue is dispensable in normal mouse development, but that the sensitivity of erythroid progenitors to Epo is regulated by the expression of EpoR.
Erythropoietin and the Peripheral Nervous System
Production and Localization in PNS
Erythropoietin and its receptor are also present in the peripheral nervous system, specifically in the bodies and axons of ganglions in the dorsal root, and at increased levels in Schwann cells after peripheral nerve injury (Campana 2001). The distribution of EpoR is different than Epo, specifically in some neuronal cell bodies in the dorsal root ganglion, endothelial cells, and Schwann cells of normal nerves. Most importantly, experiments with immunostaining revealed that the distribution and concentration of EpoR on Schwann cells doesn’t change after peripheral nerve injury. This is in agreement with research that showed Epo is up-regulated according to mRNA expression in astrocytes and hypoxia-induced neurons, while EpoR is not (Bernaudin 2000). A correlation between the expression of Epo-R in ganglion cells and binding to sensory receptors in the periphery like Pacini bodies and neuromuscular spindles suggests that Epo-R is related to touch regulation (Lykissas 2007).
Erythropoietin in Peripheral Nerve Injury
Site of Injury
After nerve injury, the increased production of Epo may induce activation of certain cellular pathways, while the concentration of EpoR doesn’t change. In Schwann cells, increased erythropoietin levels may stimulate Schwann cell proliferation via JAK2 and ERK/MAP kinase activation to be explained later. Similar to stimulation of red blood cell precursor cells (erythrogenesis), erythropoietin stimulates non-differentiated Schwann cells to proliferate (Lykissas 2007)
Although the mechanism is unclear, it is apparent that erythropoietin has anti-apoptotic action after central and peripheral nerve injury. Cross-talk between JAK2 and NF-kB signaling cascades has been demonstrated to be a possible factor in central nerve injury. Erythropoietin has also been shown to prevent axonal degeneration when produced by neighboring Schwann cells with nitrous oxide as the axonal injury signal (Keswani 2004).
Erythropoietin’s Mode of Action
Direct and Indirect Effects
Erythropoietin exerts its neuroprotective role directly by activating transmitter molecules that play a role in erythrogenesis and indirectly by restoring blood flow (Springborg 2002). Springborg and colleagues investigated the effects of a subcutaneous administration of RhEpo on cerebral blood flow autoregulation after experimental subarachnoid hemorrhage. By examining different groups of male Sprague-Dawley Rats they found that the injection of Epo after induction of hemorrhage normalized the autoregulation of cerebral blood flow, while those treated with a vehicle showed no autoregulation.
Pathway of Action
The pathway for erythropoietin in both the central and peripheral nervous systems begins with the binding of Epo to EpoR. This leads to the enzymatic phosphorylation of PI3-K and NF-kb and results in the activation of proteins that regulate nerve cell apoptosis (Chong 2002). Recent research shows that Epo activates JAK2 cascades which activate NF-kB, leading to the expression of CIAP and c-IAP2, two apoptosis-inhibiting genes. Research conducted in rat hippocampal neurons demonstrates that the protective role of Epo in hypoxia-induced cell death acts through extracellular signal-regulated kinases ERK1, ERK2 and protein kinase Akt-1/PKB (Siren 2001). They found that the action of Epo is not limited to just promoting cell survival and that the inhibition of neural apoptosis underlies short latency protective effects of Epo after brain injury. Accordingly, the neurotrophic actions may demonstrate longer-latency effects, but more research needs to be conducted on its clinical safety and effectiveness.
Pathway for Cerebral Damage and Inflammation
Additionally to the anti-apoptotic effect, Epo reduces inflammatory response during different types of cerebral injury via the NF-kB pathway (Digicaylioglou 2001). The NF-kB pathway activated by Epo/EpoR phosphorylation plays a role in regulating inflammatory and immune response, in addition to preventing apoptosis due to cellular stress (Yamamoto 2001). NF-kB proteins regulate immune response through B-lymphocyte control and T-lymphocyte proliferation. These proteins are all important for the expression of genes specific to immune and inflammatory response regulation.
As a neuroprotective agent erythropoietin has many functions: antagonizing glutamate cytotoxic action, enhancing antioxidant enzyme expression, reducing free radical production rate, and affecting neurotransmitter release. It exerts its neuroprotective effect indirectly through restoration of blood flow or directly by activating transmitter molecules in neurons that also play a role in erythrogenesis. Although apoptosis is not reversible, early intervention with neuroprotective therapeutic procedures such as erythropoietin administration may reduce the number of neurons that undergo apoptosis (Lykissas 2007).
Recombinant Human EPO Administration
The systemic administration of RhEpo has been shown to reduce dorsal root ganglion cell apoptosis (Campana 2003). While animals treated with RhEpo weren’t initially protected from mechanical allodynia after spinal nerve crush, they showed a significantly improved recovery rate compared to animals not treated with RhEpo. This RhEpo therapy increased JAK2 phosphorylation, which has been found to be a key signaling step in Epo-induced neuroprotection by an anti-apoptotic mechanism. These findings demonstrate Epo therapy as a feasible treatment of neuropathic pain by reducing the protraction of pain after nerve injury. However, more studies need to be conducted to determine the optimal time and dosage for RhEpo treatment.
Neonatal Brain Injury
In infants with poor neurodevelopment, prematurity and asphyxia are typical problems. These conditions can lead to cerebral palsy, mental retardation, and sensory impairment. However, recent research has demonstrated that high doses of recombinant erythropoietin can reduce or prevent this type of neonatal brain injury if administered early (McPherson 2007). A high rate of neuronal apoptosis is evident in the developing brain due to initial overproduction. Neurons that are electrically active and make synaptic connections survive, while those that do not undergo apoptosis. While this is a normal phenomenon, it is also known that neurons in the developing brain are at an increased risk to undergo apoptosis in response to injury. A small amount of the RhEpo can cross the blood-brain barrier and protect against hypoxic-ischemia injury. Epo treatment has also shown to preserve hemispheric brain volume 6 weeks after neonatal stroke (Gonzalez 2007). It demonstrated both neuroprotective effects and a direction towards neurogenesis in neonatal stroke without associated long-term difficulties.
Cognitive and Behavioral Effects
Systemic administration of RhEpo has also been shown to reduce lesion-associated behavioral impairment in hippocampally injured rats (Mogensen 2007). The study confirmed that Epo administration improved posttraumatic behavioral and cognitive abilities versus a saline control that experienced no improvement, although it had no detectable effect on task acquisition in non-lesioned animals. Epo is able to reduce or eliminate the consequences of mechanical injury to the hippocampus but also demonstrates possible therapeutic effects in other cognitive domains.
Epo was shown to specifically protect dopaminergic neurons, which are closely tied in to [[attention deficit hyperactivity disorder] (McPherson 2007). Specifically in mice, Epo demonstrated protective effects on nigral dopaminergic neurons in a mouse model of Parkinson’s Disease (Xue 2007). This recent experiment tested the hypothesis that RhEpo could protect dopaminergic neurons and improve the neurobehavioral outcome in a rat model of Parkinson’s Disease. The intrastriatal administration of RhEpo significantly reduced the degree of rotational asymmetry, and the RhEpo-treated rats demonstrated improvement in skilled forearm use. These experiments demonstrated that intrastriatal administration of RhEpo can protect nigral dopaminergic neurons from 6-OHDA induced cell death and improve neurobehavioral outcome in a rat model of Parkinson’s Disease.
Currently methylprednisolone (Medrol) is only pharmaceutical agent used to treat spinal cord trauma (Lykissas 2007). It is a corticosteroid that reduces damage to nerve cells and decreases inflammation near injury sites. It is typically administered within the first 8 hours after injury, but demonstrates poor results both in patients and experimental models. Some controversy has come about concerning the use of methylprednisolone because of its associated risks and poor clinical results, but it is the only medication available at this time.
If administered within a specific timeframe in experiments with erythropoietin in central nervous system, Epo has a favorable response in brain and spinal cord injuries like mechanical trauma or subarachnoid hemorrhages (Brines 2002). Brines’ research also demonstrates a therapeutic role in modulating neuronal excitability and acting as a trophic factor both in vivo and in vitro. This administration of erythropoietin functions by inhibiting the apoptosis of sensor and motor neurons via stimulation of intracellular anti-apoptotic metabolic paths. The action of erythropoietin on Schwann cells and inflammatory response after neurological trauma also points to initial stimulation of nerve regeneration after peripheral nerve injury (Lykissas 2007).
Role in Neurogenesis
Recent study by Tsai and colleagues demonstrated that erythropoietin and its receptor have an essential role in neurogenesis, specifically in post-stroke neurogenesis and in the migration of neuroblasts to areas of neural injury (Tsai 2006). They used genetics to evaluate the role of endogenous Epo and EpoR in mammalian neurogenesis. They found severe embryonic neurogenesis defects in animals that were null for Epo or EpoR genes. They also experimented with EpoR knock-down animals and found deletion of EpoR genes specific to the brain lead to a reduction in cell growth in the subventricular zone and impaired neurogenesis after stroke. This post-stroke neurogenesis was characterized by an impaired migration of neuroblasts in the peri-infarct cortex. This research agrees with the classical approach to Epo/EpoR contributions in development in that it demonstrated an Epo/EpoR requirement for embryonic neural development, adult neurogenesis, and neuron regeneration after injury. They also found that high doses of exogenous erythropoietin could demonstrate a neuroprotective role by binding to a receptor that contains the common beta receptor but lacks EpoR. These types of studies into Epo and EpoR null animals have seen and are further elucidating the neuroprotective role of Epo/EpoR in genetics and development.
A Different View: Neuroregeneration
While the neuroprotective effects of Epo administration in models of brain injury and disease have been well described, the effects of Epo on Neuroregeneration are currently being investigated. Epo administration during optic nerve transaction was used to assess the neuroprotective properties in vivo as well as demonstrate the neuroregenerative capabilities (King 2007). The intravitreal injection of Epo increased [[retinal ganglion cell somata and axon survival after transaction. A small amount of axons penetrated the transaction site and regenerated up to 1 mm into the distal nerve. In a second experiment, Epo doubled the number of retinal ganglion cell axons regenerating along a length of nerve grafted onto the retrobulbar optic nerve. This evidence of Epo as a neuroprotective and neuroregenerative agent is extremely promising for Epo as therapy in central nerve injury and repair.
Hope for the Future
Erythropoietin has shown to have a neuroprotective role in both the central and peripheral nervous system through pathways that inhibit apoptosis. It has been successful in demonstrating neuroprotective effects in many models of brain injury and in some experiments. It is also capable of influencing neuron stimulation and promoting peripheral nerve regeneration. Epo has a lot of potential uses and could provide a therapeutic answer for nervous system injury. However, more studies need to be conducted to determine the optimal time and dosage for Epo treatment.
- Neuroprotection is also a concept used in ophthalmology regarding glaucoma. The only neuroprotection currently proven in glaucoma is intraocular pressure reduction. However, there are theories that there are other possible areas of neuroprotection, such as protecting from the toxicity induced by degenerating nerve fibres from glaucoma. Cell culture models show that retinal ganglion cells can be prevented from dying by certain pharmacological treatments. However, no large clinical studies have been completed for neuroprotection in glaucoma.
- Animal studies have shown that cooling the ischemic brain can provide neuroprotection. Myron Ginsberg, MD, Director of the Cerebral Vascular Disease Research Center and Co-Director of the Neurotrauma Research Center at the University of Miami School of Medicine in Florida led studies using rats as subjects. These studies showed that by decreasing temperatures from 34 to 30 degrees Celsius, damage from global forebrain ischemia can be significantly reduced. Dr. Ginsberg said the experiments resulted in a "virtually complete preservation of pyramidal cell layer" in the CA1 hippocampus, and there was significant neuroprotection exhibited in the central and dorsal striatum, as well. Dr. Ginsberg also noted that after hypothermia treatment during focal ischemia there was a significant reduction of infarct volume.
Ashfaq Shuaib, MD, FRCPC, Director of Neurology at the University of Alberta Hospital in Edmonton, led studies which showed that post-ischemic hypothermia can provide neuroprotection, as well, given that it is of a sufficient duration and degree. 48 hours of 32 to 34 degree hypothermia of rats, initiated two and a half hours after the initiated onset of middle cerebral artery occlusion, preserved the rats' ability to retrieve food pellets in a "staircase test" of independent forelimb reaching ability.
- Bernaudin et al. Neurons and astrocytes express Epo mRNA: Oxygen-sensing mechanisms that involve the redox state of the brain. GLIA. 30: 271-278 (2000)
- Brines, M. What evidence supports use of erythropoietin as a novel neurotherapeutic? Oncology-New York. 16(9):79-89. (2002)
- Campana WM, Myers RR. Erythropoietin and erythropoieitin receptors in the peripheral nervous system: changes after nerve injury. FASEB Journal. 15:1804-1806. (2001)
- Campana WM, Myers RR. Exogenous erythropoietin protects against dorsal root ganglion apoptosis and pain following peripheral nerve injury. European Journal of Neuroscience. 18: 1497-1506. (2003)
- Celik M, Gokmen N, Erbayraktar S, Akhisaroglu M, Konakc S, Ulukus C, Genc S, Genc K, Sagiroglu E, Cerami A, and Brines M. Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury. Proceedings of the National Academy of Science. 99(4): 2258-2263. (2002)
- Chong ZZ, Kang JQ, Maiese K. Hematopoietic factor erythropoietin fosters neuroprotection through novel signal transduction cascades. Journal of Cerebral Blood Flow and Metabolism. 22:503-514. (2002)
- Digicaylioglou M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappa B signaling cascades. Nature. 412: 641-647. (2001)
- Fisher JW, Koury S, Ducey T, Mendel S. Erythropoietin (Epo) production by interstitial cells of hypoxic monkey kidneys. Br J Jaematol 95:27-32. (1996)
- Gonzalez F et al. Erythropoietin enhances long-term neuroprotection and neurogenesis in neonatal stroke. Dev Neurosci. 29:321-330. (2007)
- Grimm C, Wenzel A, Groszer M, Mayser H, Seeliger M, Samardzija M, Bauer C, Gassman M, and Reme CE. Hif-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration. Nature Medicine 8(7):718-724. (2002)
- Hoke, A, and Hoke AE. Erythropoietin and the Nervous System: Novel Therapeutic Options for Neuroprotection. Published by Springer. (2006)
- Jacobson LO, Goldwasser E, Fried W, Plzak L. Role of the kidney in erythropoiesis. Nature 179:633. (1957)
- Jelkmann, Wolfgang. Erythropoietin after a century of research: younger than ever European Journal of Haematology 78 (3), 183–205. (2007)
- Juul SE, Anderson DK, Li Y, Christensen RD. Erythropoietin and erythropoietin receptor in the developing human central nervous system. Pediatr Res 43:40-49 (1998)
- Keswani et al. A novel endogenous erythropoietin mediated pathway prevents axonal degradation. Ann Neurology. 56: 815-826. (2004)
- King CE et al. Erythropoietin is both neuroprotective and neuroregenerative following optic nerve transaction. Experimental Neurology. 205:48-55. (2007)
- Lin FK et al. Cloning and Expression of the Human Erythropoietin Gene. Proceedings of the National Academy of Science. 82(22):7580-7584. (1985)
- Lykissas et al. The role of erythropoietin in central and peripheral nerve injury. Clincal Neurology and Neurosurgery. 109: 649-644 (2007).
- Macdougall, IC. Novel erythropoiesis stimulating protein. Semin Nephrology 20:375-381. (2000)
- McPherson RJ, and Juul SE. Recent Trends in Erythropoietin-mediated Neuroprotection. Int. J. Devl Neuroscience. doi:10.1016/j.ijdevneu.2007.08.012 (2007)
- Mogensen J et al. Erythropoietin improves spatial delayed alternation in a T-maze in fimbria-fornix transected rats. Behav Brain Res. doi:10.1016/j.bbr.2007.08.009. (2007)
- Morishita E, Masuda S, Nagao M, Yasuda Y, Sasaki R. Erythropoietin receptor is expressed in rat hppocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience 76(1): 105-116. (1997)
- Siren et al. Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proceedings of the National Academy of Sciences. 98(7): 4044-4049. (2001)
- Springborg JB et al. A single subcutaneous bolus of erythropoietin normalizes cerebral blood flow autoregulation after subarachnoid hemorrhage in rats. Br J Pharmacol. 135: 823-829. (2002)
- Suzuki et al. Erythroid-specific expression of the erythropoietin receptor rescued its null mutant mice from lethality. Blood. 100(7): 2279-2288. (2002)
- Tsai, et al. A critical role of erythropoietin receptor in neurogenesis and post-stroke recovery. The Journal of Neuroscience. 26(4): 1269-1274. (2006)
- Xue YQ, Zhao LR, Guo WP, and Duan WM. Intrastriatal administration of erythropoietin protects dopaminergic neurons and improves neurobehavioral outcome in a rat model of Parkinson’s Disease. Neuroscience. 146: 1245-1258. (2007)
- Yamamoto Y, Gaynor RB. Role of the NF-kB pathway in the pathogenesis of human disease states. Current Molecular Medicine. 1: 287-296. (2001)
- Yu et al. Erythropoietin receptor signaling is required for normal brain development. Development. 129: 505-516. (2002)
- Clark, D., Dedova, I. Cordwell, S., Matsumoto, I. (2006). A proteome analysis of the anterior cingulate cortex gray matter in schizophrenia. Molecular Psychiatry, 11, 459–470.