Erythropoeitin: A Future Treatment for Traumatic Brain Injury?

Erythropoietin (EPO), first characterized in 1906, is most notably known for its role in the regulation of hematopoeisis. It was later discorvered that this protein (a member of cytokine superfamily 1) was produced by fibroblasts within the kidney in response to tissue hypoxia via hypoxia-induced transcription factor (HIF). Fast forward several decades later and it began to be discovered that not only was EPO produced by other tissues throughout the body (accounting for as much as 15-25% of production) but it also exerted effects on many tissues throughout the body. This includes the brain which has been shown to increase expression of EPO as much as 100 times when astrocytes are exposed to hypoxic conditions. EPO has been shown to cross the blood brain barrier in order to exert its effects. Many of the effects that EPO has in the brain are considered desirable in response to injury. Research over the last 15 years has indicated a potential role for EPO as a therapeutic in multiple brain diseases given its neuroprotective properties.[1] The role that EPO plays in protecting the brain from Traumatic Brain Injury (TBI) will be analyzed with an emphasis on animal models. The findings in many of the classical animals models will be discussed, as well as a look at some of the mechanisms involved, some preliminary findings from clinical trials using EPO, and finally what some of the new animal models can tell us about EPO dosing.

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Section 1: Animal Models


1.1 Cognition and Motor Function


Animal models can be used to study the effects that a TBI, or a TBI like brain injury have on memory and cognition. In humans cognitive impairment, and memory deficit have been observed following a TBI. The duration of symptoms may last a few days, or be permanent depending on severity. Animal models have demonstrated the same effects. Using tests like the Morris Water Maze, spatial memory and learning can be tracked following TBI. Remarkably one of the effects that experimenters have seen with the administration of EPO to mice and rats following injury is a decrease in the cognitive impairment, and memory loss, when compared to control animals. It should be noted that different animal models may have used different protocols for inducing TBI. In addition there are differences in the type of EPO isoforms used, the dosing of EPO, as well as the timing of EPO administration. Due to space constraints these differences, and their impact, will not be mentioned in the animal model section, however they will be discussed briefly in section 4.

Using a cryogenic injury model Grasso et. al. found that rats given recombinant human EPO had reduced cognitive deficit as demonstrated by the Morris Water Maze. Treated rats improved much faster than control rats, and no different than sham-injury rats. In addition treated rats swam faster than their control counterparts.[2]
xiongratlearning.jpg
Figure from Xiong et. al. showing fewer footfaults and greater spatial memory in rats treated with EPO. Click to enlarge

Similar results were obtained by Xiong et al using a TBI mouse model. They also used the Morris Water Maze to asses spatial learning. In addition it was found that treated with EPO had a significantly smaller increase in contralateral (to the injury) forelimb, and hindlimb footfaults following TBI, indicative of maintained sensorimotor function.[3]

Lu et. al. performed a similar experiment, however when assessing their rat TBI model for spatial learning they began morris water maze **training before the injury. They found that following the injury all of the non-sham rats immediately had trouble staying in the correct quadrant, as though they had forgotten what was previously learnt. The mice treated with EPO however, improved much faster over the following days.[4]

Click Here for more information on spatial learning and the morris water maze

Yatsiv et al. used a different method to asses their TBI mouse
model. They used the NSS (neurological severity score) scale to asses motor function, alertness, and behaviour defecits. The test works by assessing function on 10 different tasks, and a subject will be given a score of either 0, or 1, depending on their ability to complete the task. The number of failed tasks is summed so that there is a maximum score of 10 (very poor), and a minimum of 0 (indicating no cognitive deficits).
Injured rats had a large jump in their score following injury, however rats treated with EPO had a much faster declining score. In addition an object recognition test was used to asses normal behaviour. Rats are naturally exploratory animals, and when presented with a new object they tend to spend a significant amount of time exploring the new object. Therefore, when a rat spends less time exploring a novel object, this is seen as atypical, and is indicative of cognitive impairment. It was found that following TBI, injured mice spent less time exploring a new object when it was introduced to them, however this effect was smaller in the mice treated with EPO.[5]



1.2 Edema


verdonckedema.jpg
Figure from Verdonck et. all showing improved ADC with EPO administration. (Click to enlarge)

Following a TBI a number of cellular and molecular changes occur that result in cerebral edema characterized by an increased water content in the brain. This can lead to further damage through the increase in intracranial pressure, as well as impairing blood perfusion. There are two types of edema that occur following a TBI. Vasogenic edema occurs as a results of a breakdown of the blood brain barrier (BBB).[See mechanism in section 2.4] Cytotoxic edema also occurs as a result of osmotic imbalances and chemical dysregulation. Increased sodium influx causes disturbances in what is called the pump-leak-equilibrium, leading to the swelling of both neurons and glia, which can impair their function. This can also lead to ischemia of the brain tissue surrounding the lesion site.[6] Several animal models have been studied that suggest EPO may be effective in reducing post TBI edema.

Most notably is a study by Verdonck et. al. that took a close look at edema in rhEPO treated rats using an impact-acceleration TBI model. They found that rats administered with EPO 30 minutes after injury had improved tissue perfusion as seen be an increased apparent diffusion coefficient (ADC) in both the neocortex and caudaputamen. T1 mapping via MRI was used to asses brain water content (BWC). Treated rats had an attenuated increase in BWC following TBI when compared to control rats in the neocortex and caudaputamen. Moreover, the EPO treated TBI rats showed no significant difference to the sham operated group.[7]
Additional studies have also demonstrated decrease edema with EPO treatment, as well a decreased BWC on the side of the brain ipsilateral to injury.[2][8]



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chenapop.jpg
Figure from Chen. et. al. showing immunostaining and quantification of apoptosis in rats. It demonstrates a reduction in cell death for rats treated with EPO. (Click to enlarge)
1.3 Lesion Size and Cell Loss

When TBI occurs there is cell death as a result of apoptosis and necrosis resulting from physical cellular damage and cytotoxic effects. Cell death reaches its peak at 24 hours and can last for 14 days as a result of secondary injury.[3] The area of damage tissue is known as a brain lesion. Greater cell death, and toxicity is correlated with a larger lesion area.
Xiong et. al. found that cell loss in the DG was reduced by half in rats treated with EPO. Grasso et. al. observed a similar value, with a reduction in lesion size (seen on the parietal cortex) by 63% when tissue was stained with TTC (a marker for necrosis).[3] TUNEL staining has been used in multiple animal models. It has been demonstrate that when treated with EPO, there are fewer TUNEL stained cells which indicates a reduction in apoptosis. [8][9] [10]






1.4 Neurogenesis

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Figure from Zhang et. al. showing how Ara-C and EPO affect neurogenesis, and how neurogenesis relates to spatial memory. (Click to enlarge)

Until the 1990s it was believed that in mammals, following development, there was a fixed number of neurons in the brain, and throughout adulthood no new neurons were made. However, It is now well understood that neurogenesis takes place in the adult mammalian brain, particularly in he Dentate Gyrus (DG) subgranular zone, and the subventricular zone within the forebrain.[9] This process has been most notably implicated in memory formation and consolidation within the hippocampus, particularly because this is one of the primary regions where neurogenesis is known to take place.

Consequently, when there is damage to the brain, in particular when there is inflammation, the rate of neurogenesis decreases, which can be seen in TBI. Two of the animal experiments previously mentioned examined whether or not EPO has the potential to attenuate the decline in neurogenesis. Xiong and Lu injected rats with BrDU, and NeuN, and examined the staining patterns within the DG. They found that there was an increase in co-labeled NeuN and BrDU cells among rats that were treated with EPO, indicative of a greater amount of neurogenesis.[4][5]

A more in depth examination on the role of EPO in neurogenesis was performed by Zhang et. al. Not only did they examine the ability of EPO to restore neurogenesis following TBI but they designed an experiment that attempted to relate this directly to spatial memory by giving another group of rats a mitotic inhibitor in an attempt to intentionaly inhibit neurogenesis. Folliwng TBI a group of rats was treated with Cytosine-b-D-arabinofuranoside (Ara-C), and another was treated with bother EPO and Ara-C. Rats were stained for BrDU and NeuN possitive cells in the DG as well as assessed using the Morris Water Maze. The results of this study helped tie EPO mediated neurogenesis as a potential mechanism for enhanced spatial memory following TBI, as opposed to an unrelated consequence.[9]


***Note: More recent animal experiments have been conducted (since 2010) and have shown many of the same findings as the above studies. These experiments will be discussed in Section 4.


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Section 2: Cellular Mechanisms


lucidopath.jpg
Diagram from Ponce et. al. Detailing some of the cellular pathways involved in EPO signalling. (Click to enlarge)

The way in which EPO leads to many of the benefits seen in animal models is not entirely understood. However a number of experiments have been done to outline some of the molecular mechanisms that underlie many of the desired effects of EPO administration following TBI. Many of these mechanisms attenuate secondary injury that occur following TBI. These mechanisms of injury are connected, and should not be viewed as entirely separate events, but rather a few samples from the overall picture of neuronal damage resulting from TBI.
EPO primarily exerts its effects through the EPO receptor (EPOR), which is expressed on neurons, astrocytes, and endothelial cells. Endogenous EPO is also produced by a number of different cell types in the brain. It should be noted however that there is also some evidence to suggest EPO mediated neuroprotection via EPOR independent mechanisms. (See 2.6)
The binding of EPO to its receptor initiates receptor dimerization and the recruitment of JAK-2. The EPOR phosphorylates and activates JAK-2 which in turn activates a number of signaling cascades. These cascades include STAT5, PI3-K, NFKB, and RAS. The importance of these pathways has been demonstrated in vivo with the inhibition of JAK-2 and PI3K.[1] The inhibition of these proteins has been shown to prevent EPO mediated neuroprotection.[11] The many arms of the EPOR signaling cascades and the large number of associated proteins helps elucidate the ability of EPO mediated signaling to have so many physiologic effects.

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2.1 Reduced Oxidative Damage


Due to the metabolic activity as well as biochemical nature of neurons, they are particularily vulnerable to the damaging effects of reactive oxygen species (ROS). This is due in part to their metabolic nature (pathways involved in neurotransmitter production and degredation), low levels of antioxidant proteins, as well as high concentration of poly-unsaturated fatty acids (PUFA) in their membrane. ROS can cause peroxidation of PUFAs at the level of the plasma membrane which can affect membrane function and alter cellular activity as a consequence.[12] Because proper neuronal function is so heavily dependent on membrane properties, the maintenance of membrane function is critical. In vitro and In vivo studies have demonstrated that following TBI ROS levels increase in the brain, and can lead to PUFA peroxidation and oxidative damage.[13] [14] it is thought, and has been demonstrated that EPO may attenuate oxidative damage, and that this may be one of the mechanisms behind its neuroprotective effects. The ability of EPO to attenuate oxidative damage may come from its ability to increase expression of antioxidant enzymes such as glutathione peroxidase.[15]

2.2 Anti-Apoptosis


It is thought that inhibition of apoptosis may be one of the strongest mechansisms behind the beneficial effects of EPO. In particular it may be responsible for the reduced lesion size. It is well understood that following TBI there is both an upregulation and activation of several caspases involved in apoptotic pathways.[1] The ability of EPO to reduce cell death may be in part to an increase of antipoptotic gene expression, and a downregulation of pro-apoptotic genes downstream from the EPOR signaling cascades. For example the apoptotic gene Bax was found to be downregulated following the administration of EPO.[16] Conversely the antiapoptotic proteins bcl-2, XIAP, c-IAP2, and bcl-xL have been found to be upregulated.[10][17] In another experiment it was found that EPO increased SIRT1 (silent mating type information regulator 2 homolog 1) nuclear trafficking. This lead to anti-apoptotic signalling via the activation of Akt1 and FoxO3a, and the prevention of mitochondrial caspase activation, and cytochrome c leakage.[18]

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2.3 Anti-Inflammation


Apoptosis is very closely tied to inflammation given that it can be triggered by several inflammatory cytokines produced by cells of the immune system. Inflammation leads to both cell death and BBB breakdown. Animal studies have demonstrated that EPO may dampen the production of multiple inflammatory cytokines. Chen et. al. found that EPO administration following TBI in rats attenuated the inflammatory response by lowering serum levels of NFkB, TNFalpha, IL-6, and ICAM-1.[8] Similar results were seen by Bian et. al. who found decreased serum S100B protein and IL-6 levels in rats treated with EPO.[19]

2.4 Blood Brain Barrier Integrity


One of the consequences of inflammation and cell damage due to ROS, is the breakdown of the blood brain barrier (BBB). This can lead to a further increase inflammation through the passage of immune cells and inflammatory cytokines through the endothelial wall. In addition, an increase in the leakage of plasma leads to vasogenic cerebral edema. The BBB can be influenced by EPO via the expression of EPOR in cerebral endothelial cells.[20] Grasso et. al. demonstrated the ability of EPO to maintain the integrity the BBB through injection of evans blue. EPO reduced extraversion of evans blue by 60% in injured mice.[2] VEGF induced permeability as well as eNOS activity have also been implicated in the link between EPO and BBB function.[1]

2.5 EPO and LTP


Although not tested in relation to TBI another potential mechanism behind enhanced memory following EPO administration is its ability to augment Long-Term Potentiation (LTP). When rat hippocampal sclices were perfused with EPO they showed an increased propensity to form stronger synapses. Further more animal studies have demonstrated that EPO enhances memory and cognitive performance in rodents above baseline in a non-injury setting.[21] [22]

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2.6 Additional Mechanisms


Alternate mechanisms have also been suggested to account for an improved outcome with EPO administration. For example there is evidence to suggest that EPO may improve tissue perfusion following injury by triggering an increase in vascularization. Animal models have shown that EPO administration increases vascular density through vWF staining. Vascular remdoling occcurs through an increased recruitment of endothelial progenitor cells. This may occur via increased VEGF expression.[23] [24]
Through an alternate mechanism EPO signaling has been shown to trigger endothelial cells to secrete factors that cause the migration of neuroblasts towards the injured area. Inhibition of PI3K/Akt pathway attenuates this effect.[25]
Although the majority EPO effects are mediated by downstream signals of the EPOR, there is evidence to suggest alternative pathways. When the EPOR gene was inactivated in mice, EPO administration following TBI still showed beneficial effects.[26]
Another potential mechanism of cell death attenuated via EPO is zinc toxicity. In vivo and In vitro studies have demonstrated decreased zinc accumulation following TBI with EPO administration.[27]


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Section 3: Human Studies


Animal models have clearly indicated that EPO has strong potential as a novel therapeutic in brain injury, however its translation in to human studies is not yet complete, given the limited number of clinical trials. Clinical trials have indicated that the administration of EPO reduces mortality in patients admitted to hospital with severe TBI.[28] Another study demonstrated that EPO is neuroprotective in premature infants.[29]

There are currently clinical trials underway that are testing EPO as a treatment for patients with a TBI:

http://clinicaltrials.gov/ct2/show/NCT00987454?term=EPO+brain&rank=2
http://clinicaltrials.gov/ct2/show/NCT00313716?term=EPO+brain&rank=3
http://clinicaltrials.gov/ct2/show/NCT01239706?term=EPO+TBI&rank=3

One of the concerns regarding EPO as a therapeutic is the risk of thromboembolic complications as a result of abnormally high hematocrit due to increse erythropoeisis. Strategies to overcome this problem involve different EPO isoforms that are neuroprotective, but non-erythropoietic.[30] Other isoforms have been tested with a half life that is too short to stimulate erythropeisis.[1]



Section 4: Other Considerations In EPO Treatment


Many of the previously introduced animals models looked at the impact of EPO following TBI. However time of administration, and dose of EPO used did vary. Some of the newer animals studies regarding the use of EPO have examined these variables.

Ning et. al. looked at how the timing of EPO administration effected outcome. They found no difference in the outcome between rats given EPO at 6 hours and 24 hours. They used many of the same methods previously discussed to evaluate outcome including, morris water maze, footfaults, NSS, neurogenesis, and found improvements in all. Furthermore, in the same study they continued evaluating their animals as long as three months post TBI, which is longer than many of the other experiments (almost all lasted between 14 and 35 days). They found that even as long as three months later, rats treated with EPO were doing better.[24]
Mengfigure.jpg
Figure from Meng et. al. Demonstrating the dose dependent effects of EPO on spatial memory, NSS, and footfault frequency in TBI rats. (Click to enlarge)



The amount of EPO given is another variable that was hypothesized to effect outcome. Meng et. al. changed the EPO dosage. Rats were given either 1000, 3000, 5000, or 7000 U/Kg of body weight 24, 48, and 72 hours following TBI. It was found that increasing dosage improved outcome up until 5000 U/Kg, which was the most effective dose. Footfault frequency, NSS, spatial learning, and neurogenesis were assessed.[23] In another experiment the amount of EPO given was varied based on the number of times it was administered. EPO was administered to rats either once (on the first day), or three times (once a day for three days). In this case it was found that although both groups had a possitive outcome (compared to control), the rats given EPO three times did significantly better. Evaluation was based on spatial learning, footfault frequency, NSS, cell loss in the DG, and vWF staining.[31]


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References:
  1. ^ L.L. Ponce, et al., Erythropoietin neuroprotection with traumatic brain injury. Pathophysiology (2012).
  2. ^ Grasso, G. et al. Neuroprotection by erythropoietin administration after experimental traumatic brain injury. Brain Research 1182, 99-105 (2007).
  3. ^ Xiong, Y. et. al. Effects of erythropoietin on reducing brain damage and improving functional outcome after traumatic brain injury in mice. J neurosurg. 109: 510-521 (2008).
  4. ^ Lu, D. et al. Erythropoietin enhances neurogenesis and restores spatial memory in rats after traumatic brain injury. Journal Of Neurotrauma 22**, 1011-1017 (2005).
  5. ^ Yatsiv, I. et al. Erythropoietin is neuroprotective, improves functional recovery, and reduces neuronal apoptosis and inflammation in a rodent model of experimental closed head injury. The FASEB journal official publication of the Federation of American Societies for Experimental Biology 19, 1701-1703 (2005).
  6. ^ Unterberg, A.W., Stover, J., Kress, B. & Kiening, K.L. Edema and brain trauma. Neuroscience 129, 1021-1029 (2004).
  7. ^ Verdonck, O. et al. Erythropoietin protects from post-traumatic edema in the rat brain. Journal of cerebral blood flow and metabolism. 27, 1369-1376 (2007).
  8. ^ Chen, G. et al. Inhibitory effect on cerebral inflammatory agents that accompany traumatic brain injury in a rat model: a potential neuroprotective mechanism of recombinant human erythropoietin (rhEPO). Neuroscience Letters. 425, 177-182 (2007).
  9. ^ Zhang, Y., et al., Impact of inhibition of erythropoietin treatment-mediated neurogenesis in the dentate gyrus of the hippocampus on restoration of spatial learning after traumatic brain injury. Exp. Neurol. (2012).
  10. ^ Liao, Z.B., Zhi, X.G., Shi, Q.H. & He, Z.H. Recombinant human erythropoietin administration protects cortical neurons from traumatic brain injury in rats. European journal of neurology the official journal of the European Federation of Neurological Societies 15, 140-149 (2008).
  11. ^ F. Zhang, S. Wang, G. Cao, Y. Gao, J. Chen, Signal transducers and activators of transcription 5 contributes to erythropoietin-mediated neu-roprotection against hippocampal neuronal death after transient global cerebral ischemia, Neurobiol. Dis. 25, 45–53 (2008)
  12. ^ Ozturk, E. et al. Antioxidant properties of propofol and erythropoietin after closed head injury in rats. Progress in neuropsychopharmacology biological psychiatry 29, 922-927 (2005).
  13. ^ A. Chattopadhyay, T.D. Choudhury, D. Bandyopadhyay, A.G. Datta, Protective effect of erythropoietin on the oxidative damage of erythro- cyte membrane by hydroxyl radical, Biochem. Pharmacol. 59, 419–425. (2000).
  14. ^ Y. Wu, Y. Shang, S. Sun, R. Liu, Antioxidant effect of erythropoietin on 1-methyl-4-phenylpyridinium-induced neurotoxicity in PC12 cells, Eur. J. Pharmacol. 564, 47–56 (2007).
  15. ^ A. Kumral, S. Gonenc, O. Acikgoz, A. Sonmez, K. Genc, O. Yilmaz, N. Gokmen, N. Duman, H. Ozkan, Erythropoietin increases glutathione peroxidase enzyme activity and decreases lipid peroxidation levels in hypoxic-ischemic brain injury in neonatal rats, Biol. Neonate 87, 15–18. (2005).
  16. ^ Liao ZB, Jiang GY, Tang ZH, Zhi XG, Sun XC, Tang WY, Wu MJ. Erythropoietin can promote survival of cerebral cells by downregulating Bax gene after traumatic brain injury in rats. Neurol India. 57, 722-8 (2009).
  17. ^ Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades. Nature. 412, 641-7. (2001)
  18. ^ Hou, J., Wang, S., Shang, Y.C., Chong, Z.Z. & Maiese, K. Erythropoietin employs cell longevity pathways of SIRT1 to foster endothelial vascular integrity during oxidant stress. Current Neurovascular Research 8, 220-235 (2011).
  19. ^ Bian, X.-xing, Yuan, X.-song & Qi, C.-ping Effect of recombinant human erythropoietin on serum S100B protein and interleukin-6 levels after traumatic brain injury in the rat. Neurologia medicochirurgica 50, 361-366 (2010).
  20. ^ Martínez-Estrada, O.M. et al. Erythropoietin protects the in vitro blood-brain barrier against VEGF-induced permeability. European Journal of Neuroscience 18, 2538-2544 (2003).
  21. ^ El-Kordi, A., Radyushkin, K. & Ehrenreich, H. Erythropoietin improves operant conditioning and stability of cognitive performance in mice. BMC Biology 7, 37 (2009).
  22. ^ Adamcio, B. et al. Erythropoietin enhances hippocampal long-term potentiation and memory. BMC Biology 6, 37 (2008).
  23. ^ Meng, Y. et al. Dose-dependent neurorestorative effects of delayed treatment of traumatic brain injury with recombinant human erythropoietin in rats. Journal Of Neurosurgery 115, 550-560 (2011).
  24. ^ Ning, R. et al. Erythropoietin promotes neurovascular remodeling and long-term functional recovery in rats following traumatic brain injury. Brain Research 1384, 140-150 (2011).
  25. ^ L. Wang, Z.G. Zhang, R.L. Zhang, S.R. Gregg, A. Hozeska-Solgot, Y. LeTourneau, Y. Wang, M. Chopp, Matrix metalloproteinase 2 (MMP2) and MMP9 secreted by erythropoietin-activated endothelial cells promote neural progenitor cell migration, J. Neurosci. 26 5996–6003 (2006).
  26. ^ Xiong, Y. et al. Erythropoietin improves histological and functional outcomes after traumatic brain injury in mice in the absence of the neural erythropoietin receptor. Journal Of Neurotrauma 27, 205-215 (2010).
  27. ^ Zhu, L. et al. Erythropoietin prevents zinc accumulation and neuronal death after traumatic brain injury in rat hippocampus: in vitro and in vivo studies. Brain Research 1289, 96-105 (2009).
  28. ^ P. Talving, T. Lustenberger, L. Kobayashi, K. Inaba, G. Barmparas, B. Schnuriger, L. Lam, L.S. Chan, D. Demetriades, Erythropoiesis stimulating agent administration improves survival after severe trau- matic brain injury: a matched case control study, Ann. Surg. 251 (2010).
  29. ^ Auchère JC, Dame C, Vonthein R; et al. An approach to using recombinant erythropoietin for neuroprotection in very preterm infants. Pediatrics. 122(2):375-382 (2008).
  30. ^ M. Leist, P. Ghezzi, G. Grasso, R. Bianchi, P. Villa, M. Fratelli, C. Savino, M. Bianchi, J. Nielsen, J. Gerwien, P. Kallunki, A.K. Larsen, L. Helboe, S. Christensen, L.O. Pedersen, M. Nielsen, L. Torup, T. Sager, A. Sfacteria, S. Erbayraktar, Z. Erbayraktar, N. Gokmen, O. Yilmaz, C. Cerami-Hand, Q.W. Xie, T. Coleman, A. Cerami, M. Brines, Derivatives of erythropoietin that are tissue protective but not erythropoietic, Science 305 239–242 (2004).
  31. ^ Xiong, Y. et al. Delayed administration of erythropoietin reducing hippocampal cell loss, enhancing angiogenesis and neurogenesis, and improving functional outcome following traumatic brain injury in rats: comparison of treatment with single and triple dose. Journal Of Neurosurgery 113, 598-608 (2010).