Olfactory ensheathing cell phenotype after implantation into the lesioned spinal cord

Although olfactory ensheathing cells (OECs) are used to promote repair in the injured spinal cord, little is known of their phenotype in this environment. This study examined the effect of the injured spinal cord on OEC morphology and gene expression. For in situ experiments OECs were encapsulated in porous polymer tubes and implanted into the lesioned rat spinal cord. Adult male hooded Wistar rats (300-350g) were anaesthetised with isoflurane maintained at 2.5% with 100% 02 (0.5L/min). Lesions were made to the corticospinal tract at the level of T7-T8 and OEC-filled capsules were inserted under the dura above the lesion. After one week animals were sacrificed and the capsules retrieved. Procedures were performed in accordance with NHMRC guidelines approved by the University of Tasmania Animal Ethics Committee. Morphological characteristics were examined using scanning electron microscopy (SEM) for which implanted and un-implanted capsules were cut into halves so as the inner surfaces could be viewed. Implanted capsules were filled with rounded OECs and extracellular matrix (ECM)-like matter. Although no cells with the morphology of cultured OECs were observed these may have been obscured by the abundant ECM. In the un-implanted capsules cells with morphology typical of cultured OECs were predominant and although some rounded cells were detected. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to analyse expression of Neuregulin-1 (Nrg-1) and Nogo in cultured OECs and encapsulated OECs both in culture and after implantation. Neuregulins are known mitogens and survival factors for central nervous system (CNS) cells whilst Nogo is an axonal growth regulator. For comparison transcripts were also examined in a number of other glial cell types. Similar to astrocytes (ASTs) and fibroblasts (FBs), OECs expressed various Nrg-1 subtypes including neu Differentiation Factor (NDF), Glial Growth Factor (GGF) and Sensory and Motomeuron-derived Factor (SMDF) along with other splice variants including those containing the epidermal growth factor (EGF), a-EGF, ‚àövº-EGF and secreted domains. After implantation, OECs increased expression of NDF and secreted Neuregulin and decreased expression of the other variants. Olfactory ensheathing cells, oligodendrocytes (OLGs) Schwann cells (SCs) and ASTs all expressed Nogo-A, -B and -ABC. OECs and OLGs were also immunopositive for Nogo-A protein. Unlike OLGs, OECs, SCs and ASTs expressed mRNA for the Nogo-66 receptor (NgR) although the protein could not be detected by immunocytochemistry in OECs. Implantation of OECs resulted in an increase in Nogo-A and -B and a decrease in Nogo-ABC and the NgR. Taken together these results show that OEC phenotype in the lesion environment is different from their characteristic profile in culture.