Neuroprotective effect of erythropoietin in amyotrophic lateral sclerosis (ALS) model in vitro. Ultrastructural study

Introduction
Erythropoietin is the well known primary regulator of erythropoiesis, being produced by the kidney in response to hypoxic stress; it stimulates bone marrow to produce erythrocytes. For last years however, its role as cytokine involved in many physiological processes out of the bone marrow has been documented [27]. There is much evidence that EPO exerts neurotrophic and neuroprotective abilities indepen­dently of its erythropoietic action [6].
Amyotrophic lateral sclerosis (ALS) is one of the neurodegenerative diseases of upper and lower motoneurons, leading to death usually within 3-5 years, mainly because of respiratory insufficiency and hypoxemia. There is no efficient, safety and well tolerated drug discovered till now, which can stop the progression of the disease. That is why many experimental and clinical trials have been performed for years trying to find out the real pathomechanisms of the disease and secondly to reach any mediators which could potentially effect on the course of the degenerative process [23]. Multifactoral pathogenic mechanisms, including inflammation, attenuated survival signals and enhanced death signals are sugge­sted to be involved in ALS. Glutamate excitotoxicity is the well-documented pathogenic factor of neurodege­neration that is considered to contribute to the deve­lopment of neuronal cell death in ALS.
EPO is suggested to be one of compounds, that might play a potential neuroprotective role in ALS. There are many data showing that EPO helps neurons, exposed to damaging agents, to survive. It is especially involved in apoptotic mechanism of cellular death. As a neuroprotective agent, erythropoietin has many functions: antagonizing glutamate cytotoxic action, enhancing antioxidant enzyme expression, reducing free radicals production rate and affecting neurotransmitter release. It works indirectly through restoration of blood flow or directly by activating transmitter molecules in neurons. Although apoptosis is not reversible, early intervention with neuroprotective therapeutic procedures such as erythropoietin administration may reduce the number of neurons that undergo this mode of cell death [12,19]. Previous studies have shown that EPO exerts the neuroprotective effect as preconditioning and post injury adding agent. In contrast to its antiapoptotic effect, EPO has not shown any activity in preventing necrosis in the nervous system [29].
The exact mechanism leading to progressive motor neurons (MNs) loss in the course of ALS remains unclear, but glutamate-mediated mechanism is thought to be responsible. Our previous ultrastructural studies performed on a model of ALS in vitro, based on chronic glutamate excitotoxicity, revealed the coexistence of neuronal degeneration and astroglial abnormalities and suggested an active role of astrocytes in MNs degenerations [22]. The stu­dies evidenced also subset of morphological features characteristic to different modes of MNs death, including apoptotic, autophagic and necrotic dege­neration [20,21].
The aim of this study was to determine the effect of erythropoietin on ultrastructural characteristics of the MNs in organotypic cultures of rat lumbar spinal cord chronically exposed to specific glutamate uptake blocker DL-threo-beta-hydroxyaspartate (THA), pretreated with erythropoietin.

Material and methods
The study was performed on organotypic cultures prepared from lumbar spinal cord obtained from 8-day-old rat pups. The explants were placed on collagen-coated cover glasses with two drops of nutrient medium (consisting of 25% inactivated fetal bovine serum and 75% Dulbeco Modified Eagle’s Medium supplemented with glucose to a final concentration of 600mg% and with antibiotics), sealed into Maximow double assemblies and kept at 36.6°C.
On the 10-14th in vitro (DIV), the well-differentia­ted cultures were incubated with 100 M of specific glutamate uptake blocker, DL-threo-beta-hydroxy­aspartate (THA, Sigma) and erythropoietin in three experimental groups: 1) group incubated with 5 U/ml of EPO, 2) cultures treated with 100 M of THA, 3) cultures incubated with medium containing THA but pretreated with EPO (5 U/ml).
After 24 hours, 3, 5, 7, 9 and 14 days post exposition the cultures were processed for electron microscope. They were fixed in a mixture containing 0.8% formaldehyde and 2.5% glutaraldehyde for 1 hour, postfixed in 15 osmium tetroxide, dehydrated in alcohols in graded concentrations and embedded in EPON 812. Ultrathin sections were counterstained with uranylacetate and lead citrate and examined in JEOL 1200EX electron microscope.

Results
Spinal cord cultures exposed to EPO alone, displayed quite well preserved perikarya and cellular processes of MNs as well as astroglial cells, likewise the spinal cord cultures maintained in medium without additional compounds (Figs. 1 and 2). Normal appearance of MNs was characterized by round nucleus with evenly dispersed nuclear chromatin, usually prominent nucleolus and large perikaryal cytoplasm with rich granular endoplasmic reticulum, typically arranged in parallel profiles, well maintained mitochondria and Golgi apparatus.
The cultures treated with THA showed early changes even 3 hours after THA exposure. The MNs showed dispersion of rough endoplasmic reticulum, peripheral swelling and slight microvacuolization of perikaryal cytoplasm (Fig. 3). Swollen cytoplasm of postsynaptic dendrites was also observed. At 3-7 days after THA exposure there were numerous damaged MNs, exhibiting predominantly ultrastructural features of necrotic and apoptotic mode of cell death (Fig. 4). Later, 7-9 days after THA exposure, some MNs showed autophagic degenerative changes within the cytoplasm (Fig. 5). Advanced degeneration of MNs was accompanied by numerous scattered membrane-bound apoptotic bodies and fragments of degenerative cellular debris, ingested by cytoplasm of glial and phagocytic cells (Figs. 6 and 7). The cultures exposed to THA and pretreated with EPO uncommonly exhibited damaged MNs during early period of observation and increase of delayed necrosis of MNs at 7-9 day of experiment. The majority of MNs were well preserved in cultures examined at 3 hour (Fig. 8), 3 day (Fig. 9) and 5 day after EPO + THA (Fig. 10). Many MNs showed normal ultrastructural appearance of nuclei and regular arrangement of long profiles of granular endoplasmic reticulum in the cytoplasm. Mostly observed slight changes consisted of membrane-bound vacuoles located in peripheral part of cytoplasm or fine vesicular changes. The majority of glial cells and cellular processes in neuropil were also well preserved (Fig. 11). Occasionally, advanced swelling of cytoplasm and/or necrotic or autophagic type of changes were observed in individual neuronal cells (Fig. 12). At 7-9 days after EPO + THA treatment, the typical postsynaptic necrotic changes of neuronal cells occurred (Figs. 13 and 14) and were more common than during earlier period of observation. At the same time, many postsynaptic dendrites and MNs showed undamaged ultrastructure (Figs. 15 and 16). During the whole period of observation, MNs with ultrastructural characteristics of apoptotic changes were not occurred.

Discussion
The term neuroprotection refers to mechanisms which protect neurons from degeneration in various types of brain injury or chronic neurodegenerative diseases. The real mechanism of many neurodege­nerative processes remains still unknown, but one of them seems to be related with the glutamate excitotoxicity.
Previous studies have suggested that EPO might protect neurons from glutamate toxicity in vitro [25]. Erythropoietin is a chemokine hormone that is widely distributed throughout the body. EPO and its receptors (EPOR) are also present in the nervous system [7]. It plays a very important role in CNS as potential neuroprotective agent acting via direct modulation of neuronal excitability and as trophic factor for neurons both in vivo and in vitro in variety of physiological and pathological states of the nervous system.
Preclinical findings show, that EPO synthesis is triggered in response to cellular hypoxia. The erythropoietin administration occurred to protect nerve cells from hypoxia-induced glutamate toxicity [5,25] induced by many other agents as excitotoxins, glucose deprivation, kainate, serum deprivation, anoxia, NO, NMDA, glutamate and chemical hypoxia. It was shown that neurons within the penumbra would undergo apoptosis unless exposed to EPO within 3 hours after injury [30]. The expression of EPO and EPOR is highly induced by various stressors in both, animal models and human diseases [11]. It has been documented that EPO administration is also associa­ted with marked decrease in proinflammatory cytokines within hemisphere undergoing infarction. The important role of EPO in reducing immune response was established on a model of experimental autoimmune encephalomyelitis [2]. EPO has been also shown to reduce injury and enhance recovery in compression injuries of peripheral nerves [8,9,13,28]. Moreover, erythropoietin improves hippocampus dependent memory by modulating plasticity, synaptic connectivity and activity of memory-related neuronal networks [1,10].
The mechanisms of action of EPO and other potentially neuroprotective agents were identified on different, molecular, cellular and clinical levels [24]. The neuroprotective effect of EPO has been demonstrated in different animal models [26] and preclinical studies of certain nervous system diseases. This chemokine hormone might inhibit apoptosis of neurons in spinal cultures induced by kainate [31]. Systemically delivered recombinant human EPO (rhEPO) can cross blood brain barrier and inhibit neuronal death and inflammatory processes. The mechanism of neuroprotective action of EPO seems to be via stimulation of neuronal function and viability via activation of Ca2+ channels [16].
Recently, the treatment with erythropoietin is proposed in cerebrovascular events and different neurodegenerative diseases or chemotherapy-induced peripheral neuropathy [4]. EPO has been tested in clinical trial in aspect of its potential effect in stroke, schizophrenia and ALS.
Recombinant human EPO (rhEPO) has been shown to be relatively safe drug, as many patients have received it over the last decade for treatment of anemia. Well known side effects of EPO includes increased blood pressure and risk of thrombosis, especially in patients who are not anemic. Thus, the use of EPO in chronic diseases associated with neuronal apoptosis or neuroinflammation, such as Parkinson’s disease, Alzheimer disease or multiple sclerosis, would be possible only if the erythropoietic effect was blocked. In clinical trials the recombinant human erythropoietin was proposed to be used for treatment in patients with ALS [15,17]. This compound occurred usually well tolerated and safe. There have been also studies to access the effect of rhEPO in animal models of ALS, the results of which show that the treatment significantly prolongs the onset of clinical symptoms, preserves motor neurons, enhances survival signals and attenuates inflammatory signals in dose dependent manner [15].
This study support the evidence of the role of EPO in neuroprotection in model of ALS in vitro. Our previous ultrastructural study evidenced that THA exposition induces selective degeneration of MNs characterized by various types of morphological changes, including necrosis, apoptosis and autophagocytosis. The cultures pretreated with EPO exhibited less severe neuronal injury. The cultures exposed to EPO + THA showed inhibition of early MNs degeneration, including various mode of degenerative changes typical for THA effect. However, in later period (7-9 days) of observation the delayed necrotic changes of MNs, typical for excitotoxicity, were seen. The results of this study indicate that EPO exerts neuroprotective effect in the investigated model of chronic excitotoxicity mainly through prevention of apoptotic neuronal changes.
Our results as well as collected previous data [14,18] suggest that EPO may be promising therapeutic drug in different neurological diseases, including ALS.

Acknowledgements
The study was supported by the Ministry of Science and Education, grant No. 2 PO5A17429.

References
 1. Adamcio B, Sargin D, Stradomska A, Medrihan L, Gertler C, Theis F, Zhang M, Müller M, Hassouna I, Hannke K, Sperling S, Radyushkin K, El-Kordi A, Schulze L, Ronnenberg A, Wolf F, Brose N, Rhee JS, Zhang W, Ehrenreich H. Erythropoietin enhances hippocampal long-term potentiation and memory. BMC Biol 2008; 9: 37.
 2. Agnello D, Bigini P, Villa P, Mennini T, Cerami A, Brines ML, Ghezzi P. Erythropoietin exerts an anti-inflammatory effect on the CNS in a model of experimental autoimmune ence­phalomyelitis. Brain Res 2002; 11: 128-314.
 3. Arcasoy MO. The non-haematopoietic biological effects of erythropoietin. Br J Haematol 2008; 141: 14-31.
 4. Bianchi R, Brines M, Lauria G, Savino C, Gilardini A, Nicolini G, Rodriguez-Menendez V, Oggioni N, Canta A, Penza P, Lombardi R, Minoia C, Ronchi A, Cerami A, Ghezzi P, Cavaletti G. Protective effect of erythropoietin and its carbamylated derivative in experimental Cisplatin peripheral neurotoxicity. Clin Cancer Res 2006; 12: 2607-2612.
 5. Brines ML, Ghezzi P, Keenan S, Agnello D, de Lanerolle NC, Cerami C, Itri LM, Cerami A. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc Natl Acad Sci U S A 2000; 97: 10526-10531.
 6. Buemi M, Cavallaro E, Floccari F, Sturiale A, Aloisi C, Trimarchi M, Corica F, Frisina. The pleiotropic effects of erythropoietin in the central nervous system. J Neuropathol Exp Neurol 2003; 62: 228-236.
 7. Campana WM, Myers RR. Erythropoietin and erythropoietin receptors in the peripheral nervous system: changes after nerve injury. FASEB J 2001; 15: 1804-1806.
 8. Campana WM, Myers RR. Exogenous erythropoietin protects against dorsal root ganglion apoptosis and pain following peripheral nerve injury. Eur J Neurosci 2003; 18: 1497-1506.
 9. Celik M, Gökmen N, Erbayraktar S, Akhisaroglu M, Konakc S, Ulukus C, Genc S, Genc K, Sagiroglu E, Cerami A, Brines M. Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury. Proc Natl Acad Sci U S A 2002; 99: 2258-2263.
10. Ehrenreich H, Bartels C, Sargin D, Stawicki S, Krampe H. Recombinant human erythropoietin in the treatment of human brain disease: focus on cognition. J Ren Nutr 2008; 18: 146-153.
11. Eid T, Brines ML, Cerami A, Spencer DD, Kim JH, Schweitzer JS, Ottersen OP, de Lanerolle NC. Increased expression of erythropoietin receptor on blood vessels in the human epileptogenic hippocampus with sclerosis. J Neuropathol Exp Neurol. 2004; 63: 73-83.
12. Ghezzi P, Brines M. Erythropoietin as an antiapoptotic, tissue-protective cytokine. Cell Death Differ 2004; Suppl 1: S37-44.
13. Grasso G, Sfacteria A, Passalacqua M, Morabito A, Buemi M, Macri B, Brines ML, Tomasello F. Erythropoietin and erythropoietin receptor expression after experimental spinal cord injury encourages therapy by exogenous erythropoietin. Neurosurgery 2005; 56: 821-827.
14. Grasso G, Sfacteria A, Meli F, Passalacqua M, Fodale V, Buemi M, Giambartino F, Iacopino DG, Tomasello F. The role of erythropoietin in neuroprotection: therapeutic perspectives. Drug News Perspect 2007; 20: 315-320.
15. Koh SH, Kim Y, Kim HY, Cho GW, Kim KS, Kim SH. Recombinant human erythropoietin suppresses symptom onset and progression of G93A-SOD1 mouse model of ALS by preventing motor neuron death and inflammation. Eur J Neurosci 2007; 25: 1923-1930.
16. Koshimura K, Murakami Y, Sohmiya M, Tanaka J, Kato Y. Effects of erythropoietin on neuronal activity. J Neurochem 1999; 72: 2565-2572.
17. Lauria G, Campanella A, Filippini G, Martini A, Penza P, Maggi L, Antozzi C, Ciano C, Beretta P, Caldiroli D, Ghelma F, Ferrara G, Ghezzi P, Mantegazza R. Erythropoietin in amyotrophic lateral sclerosis: a pilot, randomized, double-blind, placebo-controlled study of safety and tolerability. Amyotroph Lateral Scler 2009; 10: 410-415.
18. Liu XB, Wang JA, Yu SP, Keogh CL, Wei L. Therapeutic strategy of erythropoietin in neurological disorders CNS Neurol Disord Drug Targets 2008; 7: 227-234.
19. Lykissas MG, Korompilias AV, Vekris MD, Mitsionis GI, Sakellariou E, Beris AE. The role of erythropoietin in central and peri­pheral nerve injury. Clin Neurol Neurosurg 2007; 109: 639-644.
20. Matyja E, Taraszewska A, Nagańska E, Rafałowska J. Autopha­gic degeneration of motor neurons in a model of slow glutamate excitotoxicity in vitro. Ultrastruct Pathol 2005; 29: 331-339.
21. Matyja E, Nagańska E, Taraszewska A, Rafałowska J. The mode of spinal motor neurons degeneration in a model of slow glutamate excitotoxicity in vitro. Folia Neuropathol 2005; 43: 7-13.
22. Matyja E, Taraszewska A, Nagańska E, Rafałowska J, Gebarow­ska J. Astroglial alterations in amyotrophic lateral sclerosis (ALS) model of slow glutamate excitotoxicity in vitro. Folia Neuropathol 2006; 44: 183-190.
23. Matyja E, Taraszewska A, Nagańska E, Grieb P, Rafałowska J. CDP-choline protects motor neurons against apoptotic changes in a model of chronic glutamate excitotoxicity in vitro. Folia Neuropathol 2008; 46: 139-148.
24. Maurer MH, Schäbitz WR, Schneider A. Old friends in new constellations – the hematopoetic growth factors G-CSF, GM-CSF, and EPO for the treatment of neurological diseases. Curr Med Chem 2008; 15: 1407-1411.
25. Morishita E, Maruda S, Nagao M, Yasuda Y. Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience 1997; 76: 105-116.
26. Sakanaka M, Wen TC, Matsuda S, Masuda S, Morishita E, Nagao M, Sasaki R. In vivo evidence that erythropoietin protects neurons from ischemic damage Proc Natl Acad Sci U S A 1998; 95: 4635-4640.
27. Sasaki R. Pleiotropic functions of erythropoietin. Intern Med 2003; 42: 142-149.
28. Sekiguchi Y, Kikuchi S, Myers RR, Campana WM. ISSLS prize winner: Erythropoietin inhibits spinal neuronal apoptosis and pain following nerve root crush. Spine (Phila Pa 1976) 2003; 28: 2577-2584.
29. Sinor AD, Greenberg DA. Erythropoietin protects cultured cortical neurons, but not astroglia, from hypoxia and AMPA toxicity. Neurosci Lett 2000; 290: 213-215.
30. Sirén AL, Fratelli M, Brines M, Goemans C, Casagrande S, Lewczuk P, Keenan S, Gleiter C, Pasquali C, Capobianco A, Mennini T, Heumann R, Cerami A, Ehrenreich H, Ghezzi P. Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proc Natl Acad Sci U S A 2001; 98: 4044-4049.
31. Yoo JY, Won YJ, Lee JH, Kim JU, Sung IY, Hwang SJ, Kim MJ, Hong HN Neuroprotective effects of erythropoietin posttreatment against kainate-induced excitotoxicity in mixed spinal cultures. J Neurosci Res 2009; 87: 150-163.