J Clin Neurol.  2009 Mar;5(1):39-45.

Expression of Erythropoietin in the Spinal Cord of Lewis Rats with Experimental Autoimmune Encephalomyelitis

• Kang SY
• Kang JH
• Choi JC
• Lee JS
• Lee CS
• Shin T

Abstract

BACKGROUND AND PURPOSE: Erythropoietin (Epo), originally recognized for its central role in erythropoiesis, has been shown to improve the outcomes in patients with various neurological disorders. The aim of this study was to elucidate the Epo expression pattern in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis (EAE) and to assess the systemic effect of Epo during the course of EAE.
METHODS
We used an EAE model induced in Lewis rats by immunization with myelin basic protein. Immunized rats were given recombinant human Epo (rhEpo) intraperitoneally at a dose of 5,000 U/kg for 7 consecutive days, either starting on day 3 post-immunization (five rats) or on the day of clinical symptom onset (score > or =1, five rats). After immunization, the rats were observed daily for clinical signs of EAE. Epo expression was investigated by Western blot analysis and immunohistochemistry.
RESULTS
Western blot analysis showed that, Epo expression was significantly elevated relative to control in the rat spinal cord during the peak stage of EAE (p<0.05), and then decreased thereafter. Immunohistochemistry demonstrated that Epo was expressed in some neurons and glial cells. Epo immunoreactivity was detected in ED1-positive macrophages and astrocytes in EAE lesions. Furthermore, we found that the intraperitoneal administration of rhEpo reduced both the disease severity and duration of paralysis in EAE rats, and reduced macrophage activity and increased Epo activity.
CONCLUSIONS
Based on these findings, we postulate that Epo expression begins to increase at the start of EAE and that rhEpo administration leads to functional recovery from EAE paralysis.

Keyword

astrocytes; erythropoietin; experimental autoimmune encephalomyelitis; macrophages

MeSH Terms

Animals
Astrocytes
Blotting, Western
Encephalomyelitis, Autoimmune, Experimental
Erythropoiesis
Erythropoietin
Humans
Immunization
Immunohistochemistry
Macrophages
Myelin Basic Protein
Nervous System Diseases
Neuroglia
Neurons
Paralysis
Rats
Spinal Cord
Erythropoietin
Myelin Basic Protein

Figure

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  Fig. 1 Western blot analysis of Epo expression in the spinal cord of rats with EAE. Spinal cord samples were collected from normal control rats, rats at the peak stage of EAE (G.3, day 12 PI), and rats during the recovery stage of EAE (R.0, day 21 PI). Representative photographs of Western blots; arrowheads indicate the expression of Epo and β-actin (upper). Semiquantitative analysis of Epo immunoreactivity in spinal cords, normalized to the intensity of β-actin expression in the same immunoblot. Epo was detected in the normal controls, and its expression was significantly increased in EAE-affected spinal cords. Data are (mean±SEM values) from three experiments (lower), *p<0.05 vs. normal controls and the recovery stage of EAE, †p<0.05 vs. normal controls. PI: post-immunization, Epo: erythropoietin, EAE: experimental autoimmune encephalomyelitis.

• 

  Fig. 2 Immunohistochemical staining of Epo in the spinal cords of normal control rats (A and B) and rats at the peak (day 12 PI)(C and D) and recovery (day 21 PI)(E and F) stages of EAE. Epo was weakly detected in some glial cells (A, arrows) and neurons (B, arrows) in the spinal cords of normal control rats. Glial cells at the peak stage of EAE showed increased immunoreactivity for Epo (C, arrows). Epo-positive inflammatory cells (D, arrowheads) and neurons (D, arrow) were detected in the parenchyma. At the recovery stage of EAE, some inflammatory cells (E, arrowhead), glial cells (E, arrows) and neurons (F, arrows) were positive for Epo. Tissue from a rat at the peak stage of EAE stained without the primary antisera showed no staining (G). Counterstaining was with hematoxylin. Scale bars=40 µm. Epo: Erythropoietin, PI: post-immunization, EAE: experimental autoimmune encephalomyelitis.

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  Fig. 3 Immunofluorescent co-localization of Epo (A and D) with ED1 (B) and anti-GFAP (E) in the spinal cords of rats at the peak stage of EAE (day 12 PI). A large amount of Epo (A, green; arrows) was immunostained in ED1-positive macrophages (B, red; arrows)(C, merge; arrows). Epo immunoreactivity (D, green; arrow) was co-localized in GFAP (E, red; arrow)-positive astrocytes (F, merge; arrow). Scale bars: A-F, 20 µm. Epo: Erythropoietin, anti-GFAP: anti-glial fibrillary acidic protein, EAE: experimental autoimmune encephalomyelitis, PI: post-immunization.

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  Fig. 4 Effect of recombinant human erythropoietin (rhEpo) therapy on the clinical course and protein expression in EAE. A: Rats were immunized with myelin basic protein and injected with saline (vehicle), rhEpo (5,000 U/kg) from day 3 PI (early group), or rhEpo from the day of clinical symptom onset (late group). Rats were scored daily for clinical signs of EAE. Data are the mean±SEM values from five animals per group. B: Western blot analysis of ED1 and Epo expressions in the Epo-treatment experiment. Spinal cord samples were collected from normal rats that were injected with saline (lane 1), rhEpo (lane 2), and from EAE-affected rats (day 15 PI) that were injected with vehicle (lane 3), rhEpo from day 3 PI (lane 4), and Epo from the day of clinical symptom onset (lane 5). *p<0.05 vs. vehicle. EAE: experimental autoimmune encephalomyelitis, PI: post-immunization.

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