| Abstract No.: | B-A2016 |
| Country: | Canada |
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| Title: | THE VALIDATION OF HEAT SHOCK PROTIEN 27, ANNEXIN-3A, AND CARBONIC ANHYDRASE-3 IN BOTH OLFACTORY TISSUES AND SCHWANN CELLS |
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| Authors/Affiliations: | 1 Laura Smithson, 1 T.G. McDonald,1 A. Jahed, 1 M.D. Kawaja;
1 Queen's University, Centre for Neuroscience, Kingston, ON, Canada |
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| Content: | Introduction: In an effort to treat spinal cord injury, which affects nearly 40,000 Canadians, researchers and clinicians have focused on olfactory ensheathing cells (OECs) as a viable cellular therapy to promote axonal regeneration. In the olfactory system, OECs wrap around small diameter unmyelinated olfactory axons as they exit the neuroepithelium, travel through the lamina propria of the olfactory mucosa, and course their way beyond the cribriform plate to reach their central targets in the olfactory bulb. Olfactory neurons are unique, because they have the ability to generate new axons into the environment of the adult mammalian central nervous system. OECs are credited with providing olfactory axons with a permissive environment for allowing this process of neuronal turnover and axonal outgrowth. By using the intrinsic properties of OECs, investigators have been implanting OECs into damaged spinal cords, in hopes of promoting axonal growth and functional recovery. However, OECs are known to be phenotypically very similar to Schwann cells (SCs). SCs also reside in the environment of the olfactory system, and migrate into the injured spinal cord; therefore, the purification and identification of OECs in vitro and following implantation, remains problematic. Discovering specific proteins that are expressed only in OECs is of significant value. Using proteomics, we have resolved 13 proteins that are expressed in embryonic rat OECs and not in adult rat Schwann cells. Previous immunohistochemical studies in our laboratory have validated the presence of 2 specific proteins (calponin and smooth muscle α-actin (SMA)) in adult rat OECs and the absence of these proteins in adult rat SCs, both in vivo and in culture. Objectives: The main aim of this study was to validate 3 additional proteins: heat shock protein 27 (hsp27), annexin-3A, and carbonic anhydrase III (CA-3). Results: Primary cultures of p75NTR-fluorescence-activated cell sorting (FACS) selected cells from the adult rat olfactory mucosa were hsp27/p75NTR-immunopositive cells, and annexin-3A/p75NTR-immunopositive, but not CA-3 immunopositive. Primary SCs cultured from the adult rat sciatic nerve expressed hsp27/p75NTR-immunopositive cells, while preliminary data showed no annexin-3A or CA-3 immunopositive staining. In the adult rat olfactory system, hsp27 displayed strong immunopositive staining of both mucosal and bulbar OECs, while annexin 3A showed weak labelling of mucosal OECs, and intense labelling of bulbar OECs. IgGs against CA-3 displayed no immunopositive staining for either mucosal or bulbar OECs. In the adult rat trigeminal nerve, hsp27 had no immunopositive labelling in SCs, while preliminary data suggests the presence of annexin-3A in adult rat sciatic nerve SCs. CA-3 displayed no immunopositive labelling of adult rat sciatic nerve SCs. Conclusion: Unfortunately, hsp27, annexin-3A, and CA-3 cannot be used for the purification or identification of OECs, since hsp27 and annexin-3A are present in both OECs and SCs, while CA-3 is absent in both OECs and SCs. Although proteomics can resolve various protein expression differences between OECs and SCs, the data reported here clearly indicate how essential it is to validate proteins |
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