Human recombinant erythropoietin improves motor function in rats with spinal cord compression myelopathy

OBJECTIVE Erythropoietin (EPO) is a clinically available hematopoietic cytokine. The aim of this study was to evaluate the effect of EPO on a rat model of cervical cord compression myelopathy and to explore the possibility of its use as a pharmacological treatment. METHODS To produce the chronic cervical cord compression model, thin polyurethane sheets were implanted under the C5-C6 laminae of rats and gradually expanded due to water absorption. In this model, motor functions significantly declined from 7 weeks after surgery. Based on the result, EPO administration was started 8 weeks after surgery. Motor function as seen with rotarod performance and grip strength was measured 16 weeks after surgery, and then motor neurons were stained with H-E and NeuN staining, and counted. Apoptotic cell death was assessed with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. To assess transfer of EPO into spinal cord tissue, the EPO level in spinal cord tissue was measured with an enzyme-linked immunosorbent assay for each group after subcutaneous injection of EPO. RESULTS High-dose EPO (5000 IU/kg) administered from 8 weeks after surgery markedly restored and maintained motor function in the Compression groups (P < 0.01). EPO significantly prevented loss of motor neurons in the anterior horn (P < 0.05) and significantly decreased the number of TUNEL-positive apoptotic cells (P < 0.05). The EPO level in spinal cord tissue was significantly higher in the High-dose EPO group than other groups. CONCLUSIONS EPO improves motor function in rats with progressive chronic compression myelopathy. EPO protects anterior horn motor neurons and inhibits neuronal cell apoptosis in spinal cord compression. The neuroprotective effects can be produced through transfer of EPO into spinal cord tissue. These findings suggest that EPO has high potential as a treatment for developing compression myelopathy.


6
19 Cilostazol, a selective type III phosphodiesterase inhibitor, prevent the onset of cervical 20 compression myelopathy [8,9]. However, functional recovery from developing 21 compression myelopathy has not been elucidated in those studies.

22
We recently confirmed that granulocyte colony-stimulating factor (G-CSF) improves 23 motor function in the progressive phase of compression myelopathy and preserves 24 anterior horn motor neurons in the rat chronic spinal cord compression model [10]. 25 However, in healthy people, G-CSF causes marked leukocytosis, which commonly 26 results in fever, arthralgia, and rarely, thromboembolism and splenomegaly [11].
27 Erythropoietin (EPO) is a physiological hematopoietic cytokine like G-CSF. EPO is a 28 30.4-kDa glycoprotein secreted from the kidney that stimulates red blood cell (RBC) 29 production (erythropoiesis) after binding to the EPO receptor in the bone marrow 30 [12]. EPO is commonly used in anemic patients undergoing chronic hemodialysis or 31 suffering from cancer and undergoing chemotherapy [13,14]. EPO is also used for 32 preoperative autologous blood donation in hematologically healthy individuals [15].
33 Therefore, EPO can often be used safely, even in elderly patients or those with critical 34 disease.

35
In addition, EPO has multifunctional tissue-protective effects, including anti-apoptotic, 7 37 decades, quite a few reports have described its neuroprotective effects in cerebral 38 infarction, brain contusion, and acute spinal cord injury (SCI) in laboratory investigations

59
60 Surgical procedure to create the chronic compression model

61
The detailed surgical procedure to create the chronic cervical compression model has 62 been described [7]. Under general anesthesia with 2% isoflurane, a midline incision was 63 made in the nuchal area, and the C3-Th1 laminae were exposed. A sheet of expandable

84
Briefly, 40 rats were allocated to two groups; Sham operation group (n = 15) and 85 Compression group (n = 25). In the Sham group, rats underwent a sham operation; the 86 polymer sheet was placed under the laminae and removed immediately. In the 87 Compression group, this polymer sheet was left in place and continued to compress the 88 spinal cord chronically (Fig 1A, B). The motor functions were evaluated once a week 89 from 1 week before surgery to 26 weeks after surgery. 232 weeks (Fig 3A).
Forelimb grip strength increased until 5 weeks after surgery and started to decrease 234 from 6 weeks in the Compression group. The strength gradually declined and 235 significantly decreased after 7 weeks compared with the Sham group (P < 0.001) (Fig   236 3B).

237
Based on these results, we decided to administer EPO beginning 8 weeks after surgery 238 as a treatment experiment. The rotarod performance of the Compression groups (Vehicle, Low-dose EPO, High-256 dose EPO groups) declined gradually from 5 weeks after surgery, and a significant 257 decrease was seen from 7 weeks compared with the Sham group (P < 0.005) as in the 258 preliminary experiments (Fig 3A).

259
After EPO administration beginning 8 weeks after surgery, rotarod performance 260 started to improve in the treatment groups (Low-dose EPO, High-dose EPO group). 267 Low-dose EPO group showed slightly improved rotarod performance, but did not show 268 significant improvement compared with the Vehicle group (Fig 4A).

269
The forelimb grip strength of the Compression groups decreased at 1 week after 270 surgery but started to recover gradually from 2 weeks after surgery. The strength of the 271 Compression group showed an improvement course equal to that of the Sham group 272 from 2 weeks after surgery and then started to decrease gradually from 7 weeks; at this 273 time, the strength was significantly decreased compared with the Sham group (P < 274 0.001) (Fig 4B).

275
After EPO administration at 8 weeks after surgery, grip strength started to improve in 276 the treatment groups (Low-dose EPO, High-dose EPO groups).

277
In the High-dose EPO group, the strength significantly improved compared with the 278 other Compression groups (Vehicle, Low-dose EPO groups) (P < 0.0001). Its effects 279 continued throughout the period of EPO administration (9 to 16 weeks after surgery), 280 although the strength gradually decreased from 4 weeks after EPO administration.

281
In contrast, the Low-dose EPO group showed a slight improvement in strength, but did 282 not show significant improvement compared with the Vehicle group (Fig 4B).

514
The detailed mechanisms of the neuroprotective effect of EPO for compression 515 myelopathy remain to be elucidated. In addition, in this study, the changes in local 516 blood flow and oxygen partial pressure in the spinal cord were not elucidated. However, 34 517 this study strongly suggests that EPO has potential for treating patients with developing 518 compression myelopathy, and may be worth reconsidering for clinical use to provide 519 both neuroprotective and hematopoietic effects. Further investigations including larger 520 randomized controlled trials with long-term follow-up surveys are required to establish 521 the clinical efficacy of EPO treatment and elucidate therapy-related adverse events.