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_v1.6 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.

Introduction

Olfactory ensheathing cells (OECs) are specialized glial cells that guide the regeneration of nonmyelinated olfactory axons from the peripheral nasal epithelium through the cribriform plate of the ethmoid bone and into the olfactory bulb. They are located in the olfactory epithelium, olfactory nerve and the outer layers of the olfactory bulb (Ramon-Cueto and Avila, 1998). The olfactory neuroepithelium undergoes continuous turnover and sends new axons into the olfactory bulb. OECs are thought to play a critical role in guiding the regrowth of olfactory neurons (Devon and Doucette, 1992). Subtypes of OECs have been described as Schwann cell-like (S-type), that can form peripheral myelin, and fibroblast-like (A-type) that can form a cellular channel through which axons can grow (Li et al., 1998, Choi and Raisman, 2005).

Several studies have demonstrated enhanced functional recovery after OEC transplantation into the injured spinal cord (Li et al., 1998, Ramon-Cueto et al., 2000, Plant et al., 2002, Verdu et al., 2003, Sasaki et al., 2004, Garcia-Alias et al., 2005). While the precise mechanism of this functional recovery is not fully understood, several mechanisms have been suggested including remyelination (Devon and Doucette, 1992, Franklin et al., 1996, Imaizumi et al., 1998, Sasaki et al., 2004), long axon tract regeneration (Li et al., 1997, Ramon-Cueto et al., 2000, Imaizumi et al., 2000), axonal sparing (Plant et al., 2002) and plasticity associated with novel polysynaptic pathways (Keyvan-Fouladi et al., 2002, Bareyre et al., 2004). In addition, recruitment of endogenous SCs (Takami et al., 2002, Boyd et al., 2004, Ramer et al., 2004) and remote inhibition of apoptosis of motor cortical neurons (Sasaki et al., 2006a) have been suggested to contribute to improvement in functional outcome in injured spinal cord after OEC transplantation.

OECs have also been considered to enhance repair of peripheral nerve fibers. The rationale is that they may provide a scaffold for the regenerating axons as well as trophic factors and directional cues (Deumens et al., 2006). Transplantation of OECs into axotomized facial nerve has been shown to enhance axonal sprouting (Guntinas-Lichius et al., 2001, Deumens et al., 2006) and to promote the recovery of vibrissae motor performance (Guntinas-Lichius et al., 2002). Choi and Raisman (2005) demonstrated that the rate of eye closure was increased following OEC transplantation in a facial nerve lesion model, but that aberrant nerve branching was not changed.

While a number of recent studies have focused on the potential role of OECs in enhancing functional outcome following peripheral nerve transection, little is known of the detailed integration of OECs into the peripheral nerve injury site. To address this issue, OECs from adult green fluorescent protein (GFP)-expressing transgenic rats were transplanted into a sciatic nerve crush lesion, which transects all axons. We report that GFP-expressing OECs survived for at least 6 months and distributed longitudinally along regenerated axons and remyelinated the axons. Furthermore, the regenerated fibers show reconstructed nodes of Ranvier with proper sodium channel organization, i.e., sodium channel Na_v1.6. Thus, transplanted GFP-expressing OECs prepared from adult olfactory bulb are able to remyelinate regenerated peripheral nerve fibers and restore proper nodal structure in injured PNS indicating that they can contribute to local nerve repair.