| ObjectivesTo investigate the effects of recombinant human erythropoietin (rhEPO) in a rat model of cervical sub-acute spinal cord compression.Methods80 Wistar rats were randomly divided into 4 groups. Rats in the Sham group (Group A, n=5) underwent surgical procedures without cervical spinal cord compression; while rats in other groups were subjected to the spinal compression process. In the Control group (Group B, n=25), rats were received an i.v. injection of 1 mL saline at day 7 post-surgery. Rats in the low-dose group (Groups C, n=25) and the high-dose group (Group D, n=25) were treated with rhEPO at 500 units/kg body-weight and 5000 units/kg, respectively, via intravenous injection at day 7 post surgery. Limb motor function was scored by Basso-Beattie-Bresnahan (BBB) standards at 3,7,14,21 and 28 days post-surgery. The distribution and quantities of EPO and its receptor (EPO-R) in the compressed segment of the spinal cord were detected by immunohistochemistry. Motor neuron apoptosis in the spinal cord was evaluated using TUNEL staining and flow cytometry at the indicated time points. Finally, IL-8, TNF-a, IL-6, and IL-1?levels in the compressed cervical spinal cord were determined by ELISA within the lesion epicenter at each time point post-surgery.ResultsSome of the rats died within 24 hours due to acute compression (none in Group A, three in Group B, two in Group C, three in Group D, all were excluded from the experiment and replaced). Paralysis caused by sub-acute compression occurred at 7 days or later and was characterized by the following symptoms:(1) The degree of body rigidity-light or just flaccid paralysis; (2) The forepaws were flexed slightly; animals could crawl and take food well; (3) The bladder was filled but had no incontinence or uroschesis; (4) Most rats became progressively bradykinetic, less active and assumed an abnormal posture.Immunohistochemical stains indicated that EPO and EPO-R were expressed in rat spinal cords under both normal and compressed conditions. While there was no dramatic changes in EPO expression at 14 days after surgery in Group B (compressed controls), significant overexpression of EPO-R was observed in the compressed controls compared to controls (Figurel C,p<0.05).In the control group (Group B), the injury of motor and sensory functions caused by the compressive injuries was graded according to the time and degree of compression. At 21 and 28 days post-surgery, the rats showed severe motor injury and defects in movement of one or two limbs. Although two rats exhibited complete paralysis at 28 days, there was no significant difference in BBB scores between 21 and 28 days in the group.In the rhEPO treatment group (Group C or D), however, rats exhibited marked improvement of motor function by day 14 (p<0.05). At 21 and 28 days, rhEPO greatly improved limb motor functions, but only a minor difference was observed between the low-dose and the high-dose rhEPO treatment groups. As expected, sham-treated rats did not exhibit motor deficits compared with the control group (mean motor score of 21;n=5).In the control group, TUNEL staining on rat spinal cord sections showed few positive cells at 7 days post-surgery. At 14 days, TUNEL-positive cells were observed in the gray matter, including the anterior and posterior horns as well as the posterior columns at the most compressed site. At 21 days, adjacent spinal cord sections showed large TUNEL-positive motor neurons within the ventral gray matter, as well as associated smaller cells of uncertain morphology. The number of TUNEL-positive neurons was the largest in the ventral-to-dorsal direction.After high-dose rhEPO treatment, compressed spinal cords were devoid of TUNEL staining; only sporadically-stained cells were observed in the anterior and posterior horns at 7 and 14 days. The number of TUNEL-positive cells surrounding motor neurons was less than that of the control group. No significant TUNEL staining was observed within the white matter even in the posterior column.In the control group, increasing numbers of apoptotic cells began to appear at 14 days, reached a peak at 21 days, and persistently presented thereafter. Both low and high doses of rhEPO treatment significantly decreased the apoptosis rate of resident cells in the compressed spines (p<0.01). Flow cytometry analysis also confirmed the therapeutic influence of rhEPO treatment at day 21 following surgery, suggesting a dramatic reduction of pro-apoptotic signaling. There was no significant difference among the experimental groups at 7 days. These results are in line with the motor scores obtained in this study.A time-dependent increase in inflammatory cytokine expression in the spinal cord was observed within the lesion epicenter by ELISA at 3,7,14,21 and 28 days after injury. MIP-2 (IL-8) was markedly over-expressed within 3 days and maintained up to 28 days. Treatment with low or high doses of rhEPO was associated with a gradual decrease in MIP-2 levels starting at 14 days post-surgery. TNF-a expression peaked at 7 days and remained at much higher levels compared with the controls for at least 21 days post-surgery. rhEPO administration prevented these increases. IL-6 also peaked at 7 days; while rhEPO reduced the maximum IL-6 levels to that of the control group. IL-1?increased during the first 3 days and then decreased gradually at 14 days post-surgery and was maintained at a lower level. rhEPO treatment again resulted a significant decrease starting at day 7.ConclusionsrhEPO administration decreased motor neuron apoptosis in the cervical spinal cord, improved motor functions and reduced the inflammatory response in a sub-acute cervical spinal cord compression model. Moreover, sustained treatment with low doses of rhEPO revealed a positive therapeutic effect. |
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