Ensheathing cells and methylprednisolone promote axonal regeneration and functional recovery in the lesioned adult rat spinal cord

Abstract

Axons fail to regenerate after spinal cord injury (SCI) in adult mammals, leading to permanent loss of function. After SCI, ensheathing cells (ECs) promote recovery in animal models, whereas methylprednisolone (MP) promotes neurological recovery in humans. In this study, the effectiveness of combining ECs and MP after SCI was investigated for the first time. After lesioning the corticospinal tract in adult rats, ECs were transplanted into the lesion, and MP was administered for 24 hr. At 6 weeks after injury, functional recovery was assessed by measuring successful performance of directed forepaw reaching (DFR), expressed as percentages. Axonal regeneration was analyzed by counting the number of corticospinal axons, anterogradely labeled with biotin dextran tetramethylrhodamine, caudal to the lesion. Lesioned control rats, receiving either no treatment or vehicle, had abortive axonal regrowth (1 mm) and poor DFR success (38 and 42%, respectively). Compared with controls, MP-treated rats had significantly more axons 7 mm caudal to the lesion, and DFR performance was significantly improved (57%). Rats that received ECs in combination with MP had significantly more axons than all other lesioned rats up to 13 mm. Successful DFR performance was significantly higher in rats with EC transplants, both without (72%) and with (78%) MP, compared with other lesioned rats. These data confirm previous reports that ECs promote axonal regeneration and functional recovery after spinal cord lesions. In addition, this research provides evidence that, when used in combination, MP and ECs improve axonal regrowth up to 13 mm caudal to the lesion at 6 weeks after injury.

Fig. 1.

DFR apparatuses and functional performance.A, A schematic of the DFR apparatus, viewed from the front, with the opening in the floor at 0.75 inches. Thecircles represent food pellets used as a reward.B, The DFR apparatus, viewed from above, with the opening in the floor at 0.75 inches, as was used for all testing procedures. C, Graph showing the successful performance of the DFR task (expressed as percentages of total attempts) when tested before surgery. As was required for inclusion in the study, the rats in all of the groups performed the DFR task successfully at least 90% of the trials. D, Graph showing DFR success for each group after surgery (expressed as percentages of total attempts). The sham group (94.86 ± 0.86%) performed significantly better than all of the other groups. There was no significant difference in DFR success between the lesion (38.38 ± 8.31%) and vehicle (42.41 ± 7.10%) groups, although both performed significantly worse than the MP group (56.66 ± 5.63%). Groups receiving ECs, both alone (71.84 ± 5.20%) and in combination with MP (78.26 ± 0668%), performed the DFR task significantly better than all other lesioned animals; however, these two groups were not significantly different from each other.

Fig. 2.

Fluorogold-labeled CST neurons. These neurons, filled with the retrograde tracer Fluorogold, are the origin of the corticospinal axons that were severed in the spinal cord at C3. Fluorogold can be seen filling the soma, axons, and dendrites of these neurons. Scale bar, 50 μm.

Fig. 3.

Data from 1, 10, and 19 mm caudal to the lesion. Representative photomicrographs of axons from the lesion, vehicle, MP, EC, MP/EC, and sham groups at each of these three distances caudal to the lesion. Axons are red because they are labeled with BDT. At 1 mm caudal to the lesion, there is no significant difference between mean number of axons of the lesion (A; 9.58 ± 5.53) and vehicle (D; 10.95 ± 3.25) groups, but there are significantly more axons in the MP group (G; 32.01 ± 3.41). The EC group (J; 43.57 ± 3.05) has significantly more axons than the MP group, and the MP/EC group (M; 55.99 ± 2.96) has significantly more axons than all other lesioned groups. At this distance only, there is no significant difference between the MP/EC and sham (P; 59.02 ± 4.01) groups. At 10 mm distal to the lesion, there is no significant difference between the lesion (B; 0.66 ± 0.59), vehicle (E; 0.73 ± 0.73), and MP (H; 5.85 ± 0.91) groups. All three of these groups have significantly fewer axons than the EC group (K; 12.35 ± 2.57), and the EC group has significantly fewer axons than the MP/EC group (N; 22.48 ± 6.00). There are significantly more axons in the sham group than in all the other groups at this distance (Q; 53.26 ± 3.88). At 19 mm, the lesion (C; 0.02 ± 0.02), vehicle (F; 0.04 ± 0.04), and MP(I; 0.18 ± 0.03) groups are still not statistically different. The EC (L; 2.18 ± 0.16) and MP/EC (O; 2.23 ± 0.20) groups have significantly more axons than all other lesioned groups, but there is no significant difference between these groups. The sham group has significantly more axons than all other groups at this distance (R; 52.19 ± 2.96). Scale bar, 50 μm.

Fig. 4.

Graphical and tabular presentation of the axonal data. Graphs of the mean number of axons for the lesion, vehicle, MP, EC, and MP/EC groups at 1 mm (A), 10 mm (B), and 19 mm (C) caudal to the lesion. Because the axon means are so high at all distances relative to the axon means for the other groups, the data for the sham group are only presented graphically at 1 mm caudal to the lesion (A). The table (D) is included to provide the complete set of data for the axon counts at the seven distances (1, 4, 7, 10, 13, 16, and 19 mm) caudal to the lesion for the three control and three experimental groups used in this study. Data are presented as mean ± SEM number of axons.

Fig. 5.

ECs colocalized with axons. Photomicrographs of BDT-labeled CST axons (red) surrounded by Cell Tracker green-labeled ECs. ECs in both the EC and MP/EC groups were at all distances, and representative images are shown from 1 mm (A and B, respectively), 7 mm (C and D), 13 mm (E andF), and 19 mm (G andH), the farthest distance analyzed. Scale bar, 50 μm.