Bone Marrow Transplantation or G-CSF Treatment Can Accelerate Recovery of Injured Spinal Cord in Mice.

Abstract

Mice, unlike rats and humans, have a self recovery mechanism of spinal cord injury. Whether the hematopoietic system is involved in this mechanism is under investigation. In this study we tested whether bone marrow cells transplanted or mobilized by a growth factor in mice with spinal cord injury, can accelerate the recovery. C57bl/6 female mice 10 to 12 weeks of age underwent spinal cord incision in an open operation. The injury was performed as a complete transection including the dura mater and the whole circumference of the cord at the T10-T11 intervertebral space with a micro scalpel (No 11). Group A mice received 200μg/kg/day G-CSF subcutaneously for 7 days, starting 24 hours after operation. Group B mice received 106 light density bone marrow cells from C576bl/6 donor mice intravenously 24 hours after operation. Control group mice received no treatment. Histological evaluation was performed at 48 hours, 1 week, 3 weeks and 5 weeks postoperatively. Paraffin embedded longitudinal samples of spinal cord were cut as serial sections. Spinal cord damage was estimated by measuring the maximum diameter of the area of axonal damage and disruption of astrocytic network using immunostaining for neurofilaments and GFAP. Antibodies against CD68 were applied to identify macrophage aggregations. All measurements were performed by morphometric photo analysis. The volume of fibroblastic infiltration was estimated using a grading system (0–7), based on Van Gieson stain for connective tissue. Functional deficits and recovery over time were evaluated by testing hind limb reflex and coordinated motor function (Kuhn and Wrathal functional tests, modified by Seki et al, 2002). All tests have been videotaped. Outcome scores at 48 hours, 1 week, 3 weeks and 5 weeks postoperatively for the control group, group A and group B mice were analyzed with the Mann-Whitney U test. 48 hours post operatively all mice in all groups were paralyzed in both hind limbs. Gradual improvement was observed in all groups. At week 3 there was a significant difference between the mean scores of functional tests for both treated groups (A and B) compared with the mean scores of the control group. Statistically significant difference (p<0,05) was observed in 5 out of 7 tests for group A and in 3 out of 7 tests for group B. Same difference between Group A mice and control group mice was observed by 5 weeks, while group B had no statistically significant difference. No animal in any of the groups had a complete recovery 5 weeks postoperatively. Spinal cord in control group mice showed a gradually increase of fibroblastic infiltration until 5 week which entirely separated the two ends of the cord. In group A and group B mice a significant decrease of fibroblastic infiltration was observed at week 5 compared with week 3. Macrophage aggregations were evident at weeks 1 and 5 but not at week 3 in all groups. In conclusion our results indicate that light density bone marrow transplanted cells or G-CSF treatment can accelerate spinal cord injured mice recovery. It is possible that this is associated with a decrease in fibroblastic infiltration of spinal cord. Macrophage aggregation may also play an important role in the mechanism of recovery in mice, while in rats a different reaction including cavitation and delayed demyelination prohibits neurological recovery.