Erythropoietin attenuates the sequels of ischaemic spinal cord injury with enhanced recruitment of CD34+ cells in mice

Abstract

Erythropoietin has been shown to promote tissue regeneration after ischaemic injury in various organs. Here, we investigated whether Erythropoietin could ameliorate ischaemic spinal cord injury in the mouse and sought an underlying mechanism. Spinal cord ischaemia was developed by cross-clamping the descending thoracic aorta for 7 or 9 min. in mice. Erythropoietin (5000 IU/kg) or saline was administrated 30 min. before aortic cross-clamping. Neurological function was assessed using the paralysis score for 7 days after the operation. Spinal cords were histologically evaluated 2 and 7 days after the operation. Immunohistochemistry was used to detect CD34(+) cells and the expression of brain-derived neurotrophic factor and vascular endothelial growth factor. Each mouse exhibited either mildly impaired function or complete paralysis at day 2. Erythropoietin-treated mice with complete paralysis demonstrated significant improvement of neurological function between day 2 and 7, compared to saline-treated mice with complete paralysis. Motor neurons in erythropoietin-treated mice were more preserved at day 7 than those in saline-treated mice with complete paralysis. CD34(+) cells in the lumbar spinal cord of erythropoietin-treated mice were more abundant at day 2 than those of saline-treated mice. Brain-derived neurotrophic factor and vascular endothelial growth factor were markedly expressed in lumbar spinal cords in erythropoietin-treated mice at day 7. Erythropoietin demonstrated neuroprotective effects in the ischaemic spinal cord, improving neurological function and attenuating motor neuron loss. These effects may have been mediated by recruited CD34(+) cells, and enhanced expression of brain-derived neurotrophic factor and vascular endothelial growth factor.

Fig 1

A mouse model of SCI was developed by cross-clamping the descending thoracic aorta through left thoracotomy. A small aneurysm clip was placed across the descending aorta just distal to the left subclavian artery (A). Schematic diagram of A (B). LAA: left atrial appendix; LSA: left subclavian artery; AA: aortic arch; Bar = 1 mm.

Fig 2

Survival rates of groups subjected to 7-min. ischaemia (A) and 9-min. ischaemia (B) are illustrated using Kaplan–Meier survival curves. 7C: control group subjected to 7-min. ischaemia; 7E: EPO group subjected to 7-min. ischaemia; 9C: control group subjected to 9-min. ischaemia; 9E: EPO group subjected to 9-min. ischaemia. Survival rates at day 2 after SCI were 70.6%, 68.8%, 73.3% and 74.3%, and 65.5%, 63.0%, 62.9% and 63.7% at day 7 in the 7C, 7E, 9C and 9E groups, respectively. There were no significant differences in survival rate between groups 7C and 7E (P= 0.797) or between groups 9C and 9E (P= 0.971), according to the Log-rank test.

Fig 3

Neurological outcome assessed by paralysis score from 3 hrs to day 2 after SCI with 7-min. ischaemia (A) and 9-min. ischaemia (C) and from day 2 to 7 after SCI with 7-min. ischaemia (B) and 9-min. ischaemia (D). 7C: control group subjected to 7-min. ischaemia; 7E: EPO group subjected to 7-min. ischaemia; 9C: control group subjected to 9-min. ischaemia; 9E: EPO group subjected to 9-min. ischaemia; mild: subgroup including mice with mild paralysis; severe: subgroup including mice with severe paralysis. There were no significant differences in paralysis score between groups 7C and 7E or between groups 9C and 9E until 2 days after SCI (A and C). However, group 7E-severe exhibited significant improvement between day 2 and 7, compared to group 7C-severe (B). A similar tendency was observed in group 9E-severe between day 2 and 7, compared to group 9C-severe (D). Paralysis scores in group 7C-mild, 7E-mild, 9C-mild and 9E-mild all returned to normal (grade 0) at day 7 (B and D). (B and D) *P < 0.05 with Bonferroni correction.

Fig 4

Representative cross sections stained with HE from the L1-2 level of the spinal cord after 7-min. SCI (A–P). An enlargement of the boxed regions is provided on the right side of each cross section. Almost normal morphology was maintained in lumbar spinal cords from group 7C-mild and 7E-mild (A–H). Normal motor neurons are distributed in ventral grey matter (block arrow heads). In contrast, ventral grey matter in the lumbar spinal cord from group 7C-severe and 7E-severe were largely affected at day 2 with ischaemic motor neurons (grey arrow heads) and many vacuolated regions (I, J, M and N). At day 7, affected regions in grey matter were replaced with a high density of inflammatory cells including microglia (K, L, O and P). Note that lesion area was limited in group 7E-severe at day 7 (O and P), compared to group 7C-severe (K and L). Scale bar = 250 μm in low-magnification images and 100 μm in high-power images. Bar graphs illustrating the number of normal motor neurons in the thoracolumbar spinal cord in each group with 7-min. ischaemia (Q) and 9-min. ischaemia (R). Motor neurons were well preserved in groups 7C-mild, 7E-mild, 9C-mild and 9E-mild, whereas the loss of motor neurons was evident in groups 7C-severe, 7E-severe, 9C-severe and 9E-severe. However, higher numbers of motor neurons were preserved in groups 7E-severe and 9E-severe at day 7, compared to groups 7C-severe and 9C-severe. (Q and R) *P < 0.05 with Bonferroni correction.

Fig 5

Representative pictures of CD34⁺ cells in ventral grey matter (A) and enlargement of boxed area (B). Blue: nuclei and red: CD34. (A) Scale bar = 25 μm; (B) Scale bar = 10 μm. The number of CD34⁺ cells in the lumbar spinal cord after 7-min. (C) and 9-min. SCI (D). CD34⁺ cells were more abundantly recruited in the spinal cord in groups 7E-severe and 9E-severe than those in groups 7C-severe and 9C-severe at day 2 (C and D, *P < 0.05 with Bonferroni correction). CD34⁺ cells were not apparent in groups 7C-mild, 7E-mild, 9C-mild and 9E-mild. Representative pictures of immunofluorescence for BDNF in ventral grey matter from groups 9E-severe (E) and 9C-severe (F) at day 7. Blue: nuclei and green: BDNF. BDNF expression in group 9E-severe (E) was more evident than that in group 9C-severe (F). Scale bar = 25 μm. Representative pictures of VEGF expression in the left ventrolateral white matter of the spinal cord from groups 9E-severe (G) and 9C-severe (H) at day 7. VEGF was detectable in the white matter of group 9E-severe and vacuolization in white matter was limited (G). In contrast, no VEGF expression and advanced vacuolization was apparent in the white matter of group 9C-severe (H). Scale bar = 50 μm.