Neuroprotection

Neuroprotection within the nervous system protects neurons from apoptosis or degeneration, for example following a brain injury or as a result of chronic neurodegenerative diseases.

Currently, there is a broad interest in how apoptosis and neuroprotection affect the brain during development and after the onset of central nervous system (CNS ) diseases (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 neuroprotection.

The following article deals with role of Erythropoietin in Neuroprotection.

Erythrogenesis
Erythropoietin (Epo) is a glycoprotein that controls erythropoiesis, or red blood cell production.

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 et al. demonstrated the presence of Epo within the spinal fluid of infants and the expression of Epo-R in the spinal cord, suggesting a role by Epo within the CNS. Further, Grimm et al. reported the detection of Epo and Epo-R expression in the mammalian retina, and proposed a potential therapy to protect photoreceptors damaged from hypoxic pretreatment.

The ability of erythropoietin to protect nerve cells from hypoxia-induced glutamate toxicity was demonstrated in experiments by Grimm et al. in 2002 and Morishita et al. in 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. The work of Morishita and his colleagues has provided evidence for the presence of Epo-R in cultured hippocampal and cerebral cortical neurons isolated from rat embryos. Epo was capable of protecting the cultured neurons from glutamate neurotoxicity after only a short exposure. It was concluded that Epo-mediated increase in intracellular calcium concentration is indicative of Epo's neuroprotective role after CNS-related hypoxia or ischemia.

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.

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. 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. 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. 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 mutants R103-E and S100-E (though S100 in Epo doesn't exist) were first reported by Leist et al. 2004 to be non-erythropoietin but retain the neuroprotective function. Also Burns et al. 2002 reported Epo with R103 mutation as a potent inhibitor of wild type Epo from binding to its receptor. Though, the viral vector expressed R103-E Epo mutant was shown to be inhibitory to the progression / development of nervous tissue damage in many models, it is not shown to recover the nervous tissue post damage. Given the associated risks, it would be foolish to administer / express Mutant as a preventive measure from neuronal injury. Hence, from a medical or commercial point of view, safe and feasible neuro-protective Epo mutants are not possible.

It should also be noted that quite a bit of research emphesis is on non erythropoietic but, neuroprotective Peptides of Erythropoietin. According to Pankratova S et al. 2010, peptide of Epo with amino acids 92-111 is neuroprotective while its erythropoietic potency is 10 fold less than the wild type.

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. 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. 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.

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.

Anti-apoptosis mechanisms
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-κB 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.

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 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-κB and results in the activation of proteins that regulate nerve cell apoptosis. Recent research shows that Epo activates JAK2 cascades which activate NF-κB, 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. 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-κB pathway. The NF-κB pathway activated by Epo/EpoR phosphorylation plays a role in regulating inflammatory and immune response, in addition to preventing apoptosis due to cellular stress. NF-κB 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.

Neuroprotective effects
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.

Recombinant human EPO administration
The systemic administration of RhEpo has been shown to reduce dorsal root ganglion cell apoptosis. 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. Hypothermia therapy for neonatal encephalopathy is a proven therapy for neonatal brain injury. However, recent research has demonstrated that high doses of recombinant erythropoietin can reduce or prevent this type of neonatal brain injury if administered early. 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. 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. 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.

Dopaminergic neurons
Epo was shown to specifically protect dopaminergic neurons, which are closely tied in to attention deficit hyperactivity disorder. Specifically in mice, Epo demonstrated protective effects on nigral dopaminergic neurons in a mouse model of Parkinson’s Disease. 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.

Current treatment
Currently methylprednisolone (Medrol) is only pharmaceutical agent used to treat spinal cord trauma. 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.

Neurotherapeutic Role
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. 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.

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. 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.

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. 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.

Research directions
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.

Glaucoma
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. Intraperitoneal injection of Epo in DBA/2J mice protected / slowed down the degeneration of Retinal ganglion cell (RGC)- L Zhong et al. 2007. A Epo with R103 mutation, expressed from IM administered Adeno Associated viral vectors, protects the retinal ganglion cells in DBA/2J and Optic nerve crush models for glaucoma in mice. '''Erythropioetin is a very potent molecule (One Unit ~ 10 ng)and is known to be extremely toxic in higher doses. Since, such a tight control of expression is impossible, genetherapy with viral vectors is not an option.''' Overexpression of Epo and Epo mutants in the eye via, viral vectors is toxic to the retina.

Therapeutic Hypothermia
Animal studies have shown that cooling the ischemic brain can provide neuroprotection. This technique's is called therapeutic hypothermia. 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. One device used to induce therapeutic hypothermia was called the Arctic Sun.