Myelination and nodal formation of regenerated peripheral nerve fibers following transplantation of acutely prepared olfactory ensheathing cells

Abstract

Transplantation of olfactory ensheathing cells (OECs) into injured spinal cord results in improved functional outcome. Mechanisms suggested to account for this functional improvement include axonal regeneration, remyelination and neuroprotection. OECs transplanted into transected peripheral nerve have been shown to modify peripheral axonal regeneration and functional outcome. However, little is known of the detailed integration of OECs at the transplantation site in peripheral nerve. To address this issue, cell populations enriched in OECs were isolated from the olfactory bulbs of adult green fluorescent protein (GFP)-expressing transgenic rats and transplanted into a sciatic nerve crush lesion which transects all axons. Five weeks to 6 months after transplantation, the nerves were studied histologically. GFP-expressing OECs survived in the lesion and distributed longitudinally across the lesion zone. The internodal regions of individual teased fibers distal to the transection site were characterized by GFP expression in the cytoplasmic and nuclear compartments of cells surrounding the axons. Immunoelectron microscopy for GFP indicated that the transplanted OECs formed peripheral type myelin. Immunostaining for sodium channel and Caspr revealed a high density of Na(v)1.6 at the newly formed nodes of Ranvier which were flanked by paranodal Caspr staining. These results indicate that transplanted OECs extensively integrate into transected peripheral nerve and form myelin on regenerated peripheral nerve fibers, and that nodes of Ranvier of these axons display proper sodium channel organization.

Figure 1

Olfactory ensheathing cell (OEC) transplantation into transected sciatic nerve. Fluorescent images of a living sciatic nerve whole mount taken immediately upon nerve extraction and desheathment at 5 weeks post transplantation (A). B. Comparable whole mount for a fixed nerve. Green fluorescent protein (GFP)-OECs are distributed longitudinally several millimeters from the injection site. Areas of increased fluorescence are the nuclei and dense cytoplasmic regions of the OECs. C1. Higher magnification image of teased, fixed nerve taken immediately upon removal at 5 weeks post transplantation. GFP-OECs associate with individual axons within the lesion site. C2. An individual living axon with putative nodes of Ranvier (boxed) and areas of concentrated fluorescence, which indicate OEC nuclei and cytoplasm. Scale bar: A=500μm, B = 25μm, C1= 11μm, C2=3.5μm

Figure 2

Distribution of OECs in injured sciatic nerve A. Confocal images of crushed sciatic nerve at 5 weeks post OEC transplantation demonstrating an abundance of GFP-OECs distributing longitudinally in the injured nerve distal to the transection. Inset. Association of GFP fluorescence with axonal neurofilament staining. B. Caspr immunostaining (blue) demonstrating paranodal regions on regenerated axons associated with GFP-OECs. The upper inset is a higher magnification of an individual nodal region. Immunostaining for sodium channel Na_v1.6 indicates a node formation on a regenerated axon (lower inset). C. Survival of GFP-OECs on the regenerated axons 6 months post-crush injury. Scale bar: A = 200μm, B = 40μm, C = 80μm, D= 13μm, A inset=13μm

Figure 3

Na_v1.6 at nodes of Ranvier of regenerated sciatic nerve fibers remyelinated by OECs. A. Na_v1.6 sodium channel is observed at Caspr-delimited nodes formed on axons associated with GFP expressing cells (A–D). Caspr-delimited nodes formed by GFP-OECs do not exhibit Na_v1.2 immunostaining (E–H). Scale bar = 10 μm.

Figure 4

Electron micrographs of anti-GFP immunoperoxidase staining of OECs 7 weeks post transplantation. A1. Reaction product can be seen in the cytoplasm of the GFP-OECs, which have formed myelin around an axon. A2. Higher magnification of boxed area in A1. B1. Another myelinated axon showing intense reaction product in the myelin-forming cell. B2. Higher magnification of boxed area in B1. Scale bar: A1=5μm, A2=1μm, B1=1μm, B2=0.5 μm