Integration of engrafted Schwann cells into injured peripheral nerve: axonal association and nodal formation on regenerated axons

Abstract

Transplantation of myelin-forming cells can remyelinate axons, but little is known of the sodium channel organization of axons myelinated by donor cells. Sciatic nerve axons of female wild type mice were transected by a crush injury and Schwann cells (SCs) from green fluorescence protein (GFP)-expressing male mice were transplanted adjacent to the crush site. The male donor cells were identified by GFP fluorescence and fluorescence in situ hybridization (FISH) for Y chromosome. In nerves of GFP-expressing mice, GFP was observed in the axoplasm and in the cytoplasmic compartments of the Schwann cells, but not in the myelin. Following transplantation of GFP-SCs into crushed nerve of wild type mice, immuno-electron microscopic analysis indicated that GFP was observed in the cytoplasmic compartments of engrafted Schwann cells which formed myelin. Nodal and paranodal regions of the axons myelinated by the GFP-SCs were identified by Na(v)1.6 sodium channel and Caspr immunostaining, respectively. Nuclear identification of the Y chromosome by FISH confirmed the donor origin of the myelin-forming cells. These results indicate that engrafted GFP-SCs participate in myelination of regenerated peripheral nerve fibers and that Na(v)1.6 sodium channel, which is the dominant sodium channel at normal nodes, is reconstituted on the regenerated axons.

Fig. 1

Morphology of dissociated GFP-expressing mouse nerve. Bright field image (A) and fluorescence (B) images of same teased axon field. (C) Nodal regions were studied by Na⁺ channel immunohistochemistry on normal GFP-expressing mouse nerve. (D) Higher magnification of immunostained sodium channel at a node of Ranvier showing more detailed nodal and internodal structure. (E) Y chromosome identification in Schwann cells of mouse peripheral nerve in a normal sciatic nerve of a male GFP mouse. The Schwann cell nuclei were stained with DAPI (blue) and the Y chromosome (pink) was visualized with FISH. Calibrations: (A and B) 40 μm; (C and D) 10 μm; (E) 20 μm.

Fig. 2

Node of Ranvier in regenerated nerve fibers following transplantation of GFP-expressing Schwann cells into crushed non-GFP-expressing mouse nerve. (A) Note the sequential myelinated segments by the transplanted GFP-expressing cells. (B) Higher power image showing node-like structure (arrow) and two adjacent regions with intense green fluorescence near nuclei. (C) Immunostaining for Na⁺ channel shows three nodal regions with short internodes. (D) FISH (pink) for Y chromosome on transplanted male Schwann cells into crushed female sciatic nerve. Nuclei are stained with DAPI (blue). (E) Sodium channel (Na_v 1.6; red) and paranodal (Caspr; blue) immunostaining showing node formation of regenerated axons myelinated by engrafted Schwann cells (confocalimage). (F) Immunoreaction product for GFP can be seen in the cytoplasm of cells forming myelin. (G) Boxed area in (F). Calibrations: (A) 60 μm; (B and D) 20 μm; (C) 30 μm; (E) 10 μm; (F) 0.3 μm: (G) 0.1 μm.