Further evidence of olfactory ensheathing glia facilitating axonal regeneration after a complete spinal cord transection

Abstract

Spinal Wistar Hannover rats injected with olfactory ensheathing glia (OEG) have been shown to recover some bipedal stepping and climbing abilities. Given the intrinsic ability of the spinal cord to regain stepping with pharmacological agents or epidural stimulation after a complete mid-thoracic transection, we asked if functional recovery after OEG injections is due to changes in the caudal stump or facilitation of functional regeneration of axons across the transection site. OEG were injected rostral and caudal to the transection site immediately after transection. Robotically assisted step training in the presence of intrathecal injections of a 5-HT_2A receptor agonist (quipazine) was used to facilitate recovery of stepping. Bipedal stepping as well as climbing abilities were tested over a 6-month period post-transection to determine any improvement in hindlimb functional due to OEG injections and/or step training. The ability for OEG to facilitate regeneration was analyzed electrophysiologically by transcranially stimulating the brainstem and recording motor evoked potentials (MEP) with chronically implanted intramuscular EMG electrodes in the soleus and tibalis anterior with and without intrathecal injections of noradrenergic, serotonergic, and glycinergic receptor antagonists. Analyses confirmed that along with improved stepping ability and increased use of the hindlimbs during climbing, only OEG rats showed recovery of MEP. In addition the MEP signals were eliminated after a re-transection of the spinal cord rostral to the original transection and were modified in the presence of receptor antagonists. These data indicate that improved hindlimb function after a complete transection was coupled with OEG-facilitated functional regeneration of axons. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Introduction

While the results of studies on the effects of olfactory ensheathing glia (OEG) in repairing an injured spinal cord have been highly variable, so have the experimental paradigms used to determine the reparative potential of these cells, differing in the type and severity of injury, mixture of cell types, duration of transplant, transplantation procedures, and parameters used to define regeneration (Frannsen et al., 2007). One of the more important questions yet to be answered conclusively is whether OEG can facilitate regeneration of axons after a complete spinal cord transection. We therefore hypothesized that OEG implanted immediately after a complete spinal cord transection can restore functional connections between the rostral and caudal spinal cord stumps. Previous studies showed improvement in hindlimb function in OEG spinal rats, but it is unclear whether this improvement was attributable to regeneration of axons and/or to reorganization of the neurons in the caudal stump (Ramón-Cueto et al., 2000, Lu et al., 2002, Keyvan-Fouladi et al., 2003; Verdú et al., 2003, García-Alías et al., 2004, Polentes et al., 2004, López-Vales et al., 2007, Kubasak et al., 2008). Some anatomical data are consistent with the presence of regeneration, but these data are not conclusive (Ramón-Cueto et al., 2000, Takeoka et al., 2009, Takeoka et al., 2010). In the present study we focused on a battery of functional (behavioral, electrophysiological, and pharmacological) assessments during 6 months post-transection and OEG injections, some of which were performed before and after re-transection of the spinal cord.

We observed motor-evoked potentials (MEP) only in OEG rats. These MEP were modified by step training and their amplitudes could be modulated by yohimbine, a NA receptor antagonist, cyproheptadine, a 5-HT₂ receptor antagonist, and/or strychnine, a glycinergic receptor antagonist. In addition, the MEP were eliminated when the spinal cord was re-transected rostral to the original transection site.