Transplantation of ciliary neurotrophic factor-expressing adult oligodendrocyte precursor cells promotes remyelination and functional recovery after spinal cord injury

Abstract

Demyelination contributes to the dysfunction after traumatic spinal cord injury (SCI). We explored whether the combination of neurotrophic factors and transplantation of adult rat spinal cord oligodendrocyte precursor cells (OPCs) could enhance remyelination and functional recovery after SCI. Ciliary neurotrophic factor (CNTF) was the most effective neurotrophic factor to promote oligodendrocyte (OL) differentiation and survival of OPCs in vitro. OPCs were infected with retroviruses expressing enhanced green fluorescent protein (EGFP) or CNTF and transplanted into the contused adult thoracic spinal cord 9 d after injury. Seven weeks after transplantation, the grafted OPCs survived and integrated into the injured spinal cord. The survival of grafted CNTF-OPCs increased fourfold compared with EGFP-OPCs. The grafted OPCs differentiated into adenomatus polyposis coli (APC(+)) OLs, and CNTF significantly increased the percentage of APC(+) OLs from grafted OPCs. Immunofluorescent and immunoelectron microscopic analyses showed that the grafted OPCs formed central myelin sheaths around the axons in the injured spinal cord. The number of OL-remyelinated axons in ventrolateral funiculus (VLF) or lateral funiculus (LF) at the injured epicenter was significantly increased in animals that received CNTF-OPC grafts compared with all other groups. Importantly, 75% of rats receiving CNTF-OPC grafts recovered transcranial magnetic motor-evoked potential and magnetic interenlargement reflex responses, indicating that conduction through the demyelinated axons in VLF or LF, respectively, was partially restored. More importantly, recovery of hindlimb locomotor function was significantly enhanced in animals receiving grafts of CNTF-OPCs. Thus, combined treatment with OPC grafts expressing CNTF can enhance remyelination and facilitate functional recovery after traumatic SCI.

Figure 1.

In vitro differentiation of adult OPCs. All purified OPCs expressed hPAP (A–C) as well as OPCs markers O4 (A), A2B5 (B), or NG2 (C). Three days after withdrawal of FGF2 and PDGFaa, OPCs constitutively differentiated into OLs expressing O1 (D). Six days after differentiation, OPCs differentiated into mature OLs expressing MBP with complex membrane sheet (E). Two weeks after cocultured with DRG neurons, adult OPCs became OLs, which formed myelin sheaths around the axons (F). Scale bars: A, E, F, 20 μm; B, C, 10 μm; D, 50 μm.

Figure 2.

CNTF promoted OL differentiation of adult OPCs in vitro. Adult OPCs were grown in basal medium containing FGF2 (5 ng/ml) and the indicated growth factor for 3 d. The effect of individual growth factor to overcome the inhibition of FGF2 to promote differentiation was defined by the percentage of O1⁺ OLs. Compared with control (A), CNTF (B) and IGF1 (C) significantly promoted differentiation, whereas other factors had no significant effect (D). Data in D represent the mean ± SD (n = 4). ^☆p < 0.05. Scale bars, 50 μm.

Figure 3.

CNTF significantly increased OL survival and maturation in vitro. Adult OPCs were differentiated for 2 d in basal medium (BM) to become O1⁺ OLs and continued to mature for 5 more days in BM plus the indicated growth factor. Without additional trophic support, most OLs underwent apoptosis (A). Addition of CNTF prevented most OLs from initiating apoptosis (B). The surviving OLs showed mature OL morphology with complex membrane sheets. Although all tested growth factors protected differentiated OLs from death, CNTF was the most effective (C). The survival of OLs was also measured by MTT (D–F). Compared with control BM (D), there was significantly more survival with all growth factors, but CNTF was the most effective (E, F). Data in C and F represent the mean ± SD (n = 4). ^☆p < 0.05. Scale bars: A, B, 25 μm; D, E, 50 μm.

Figure 4.

CNTF promoted the survival of grafted OPCs after SCI. Adult OPCs were infected with retroviruses to express EGFP or CNTF. At 2 d after infection, >70% of OPCs were infected (A). Importantly, expression of CNTF in CNTF-OPCs was significantly higher compared with uninfected or EGFP-OPCs by ELISA (B). One week after transplantation, CNTF-OPCs continued to express CNTF in the injured spinal cord (C, arrows). The ELISA showed that CNTF concentrations in the injured spinal cord from animals receiving grafted CNTF-OPCs were significantly higher than animals receiving DMEM, or EGFP-OPCs (D). The effects of CNTF expression on the long-term survival of grafted OPCs were detected by hPAP immunohistochemistry. At 2 months after transplantation, robust graft survival was observed for both CNTF- (E–G) or EGFP-OPCs (data not shown). At higher magnification, the hPAP-immunoreactive processes formed ring-like structures that were evident in cross sections (F) and sheath-like structures readily apparent in longitudinal sections (G). The survival and process elaboration from the grafted OPCs, measured by hPAP immunoreactivity, was four time greater in CNTF-OPCs compared with EGFP-OPCs (D), indicating that expression of CNTF promoted the survival of grafted OPCs after SCI. Data in B, D and H represent the mean ± SD (n = 3 in B and D and 6 in H). *p < 0.05. Scale bars: A, C, 50 μm; E, 500 μm; F, G, 20 μm.

Figure 5.

OL differentiation of transplanted OPCs after SCI. Two months after transplantation, many grafted CNTF-OPCs differentiated into APC⁺ mature OLs in the injured spinal cord (A–F). The grafted hPAP⁺ OPCs coexpressed the mature OL marker, APC, in their cytoplasm (A–C, arrows). Similarly, the grafted OPCs identified by EGFP immunoreactivity in their cytoplasm also coexpressed APC (D–F, arrows). Although p75⁺ Schwann cells were found in the contused spinal cord, Schwann cell differentiation of transplanted OPCs was not observed (G–I, arrows). The grafted OPCs also did not differentiate into GFAP⁺ astrocytes, although gliosis, shown by GFAP immunoreactivity, was obvious in the injured spinal cord. Scale bars: A–L, 20 μm.

Figure 6.

OL remyelination by grafted OPCs after SCI. The grafted OPCs differentiated into mature APC⁺ OLs (A–C, arrowheads), which formed myelin rings around NFM⁺ axons (A–C, arrows). Double staining for hPAP and MBP in cross sections further confirmed that hPAP-immunoreactive rings around NFM⁺ axons were MBP⁺ myelin (D–F, arrows). In longitudinal section, MBP⁺ myelin sheaths from the grafted OPCs were more clearly shown along the NFM⁺ axons (G–I, arrows). Scale bars: A–I, 20 μm.

Figure 7.

CNTF increased the number of OL-remyelinated axons by grafted OPCs after SCI. Immuno-EM confirmed that OLs from grafted OPCs, as shown by hPAP immunoreactivity, formed myelin on multiple axons (A). At higher magnification (the box in A is shown in B), it is evident that DAB reaction product of hPAP immunoreactivity is found with the myelin surrounding one of the axons (B, C), indicating that these myelin sheaths were derived from grafted OPCs. OL- (D, arrows) and Schwann cell-remyelinated axons (D, arrowheads) were readily distinguished in the thin plastic section of spinal cord stained with toluidine blue. The OL-remyelinated axons (E), but not SC-remyelinated axons (F), significantly increased in both VLF and LF at the injury epicenter in rats with grafts of OPCs or CNTF-OPCs. Data in E and F represent the mean ± SD (n = 6). ^★p < 0.05; ^★★p < 0.01. Scale bar: A–C, 1 μm; D, 10 μm.

Figure 8.

Electrophysiological recovery after transplantation of adult OPCs. Six of 10 animals receiving CNTF-adult OPCs showed recovery of tcMMEP responses compared with 2 of 10 in OPC-grafted rats and none in animals receiving grafts of DMEM, FBs, or CNTF-FBs (A). The latencies (B) and amplitudes (C) of recovered tcMMEP responses improved over time. The latencies at 4 and 6 weeks were significantly shorter than at 2 weeks after injury (p < 0.05) (B). The amplitudes at 6 weeks were also significantly higher than at 2 and 4 weeks after injury (p < 0.05) (C). Eight of 10 rats receiving CNTF-OPCs grafts showed recovery of MIER responses at 8 weeks after graft compared with 4 of 10 in OPC-grafted rats, and 2 of 10 in animals receiving DMEM, FBs, or CNTF-FBs (D). The latencies (E) and amplitudes (F) of the recovered MIER responses in CNTF-OPC-grafted animals were significantly longer and small, respectively, than the baseline responses before injury (all p < 0.05). Quantitative data are the mean ± SD (n = 6).

Figure 9.

Hindlimb locomotor recovery after transplantation of adult OPCs. Locomotor function, determined using the BBB locomotor score, was significantly recovered in CNTF-OPC-grafted animals from weeks 3 to 7 after injury, and also in EGFP-OPC-grafted animals from weeks 5 to 7 after injury, compared with FB- or DMEM-grafted animals (A) (data are mean ± SD; n = 10; *p < 0.05). BBB scores were closely correlated with the number of OL-remyelinated axons in both the LF (B) and VLF (C) at the injury epicenter of spinal cord.