Potential of olfactory ensheathing cells from different sources for spinal cord repair

Abstract

Spinal cord injury (SCI) induces a permanent disability in patients. To this day no curative treatment can be proposed to restore lost functions. Therefore, extensive experimental studies have been conducted to induce recovery after SCI. One of the most promising therapies is based on the use of olfactory ensheathing cells (OECs). OECs can be obtained from either the olfactory bulbs (OB-OECs) or from olfactory mucosa (OM-OECs), involving a less invasive approach for autotransplantation. However the vast majority of experimental transplantations have been focusing on OB-OECs although the OM represents a more accessible source of OECs. Importantly, the ability of OM-OECs in comparison to OB-OECs to induce spinal cord recovery in the same lesion paradigm has never been described. We here present data using a multiparametric approach, based on electrophysiological, behavioral, histological and magnetic resonance imaging experiments on the repair potential of OB-OECs and OM-OECs from either primary or purified cultures after a severe model of SCI. Our data demonstrate that transplantation of OECs obtained from OB or OM induces electrophysiological and functional recovery, reduces astrocyte reactivity and glial scar formation and improves axonal regrowth. We also show that the purification step is essential for OM-OECs while not required for OB-OECs. Altogether, our study strongly indicates that transplantation of OECs from OM represents the best benefit/risk ratio according to the safety of access of OM and the results induced by transplantations of OM-OECs. Indeed, purified OM-OECs in addition to induce recovery can integrate and survive up to 60 days into the spinal cord. Therefore, our results provide strong support for these cells as a viable therapy for SCI.

Figure 1. Experimental paradigm illustrating the timeline of the major experimental manipulations.

Cells or medium were transplanted into sectioned spinal cords (D0). 15, 30 and 60 days after surgery animals were analyzed for electrophysiological and locomotor activities (D15, D30, D60). Histological analyses were performed 60 days after transplantation for all the groups (D60) (n = 10). On additional animals of OM-purified OECs tracking of GFP+ cells were performed 30 and 60 after transplantation (D30, D60). Each time point (n = 3) (in green).

Figure 2. P75 expression of primary OB (A) and OM (C) and purified OB-OECs (B) and OM-OECs (D) cultures.

After 5 and 10 days in vitro, cell surface expression of p75 in primary OB and purified OB cultures was determined by flow cytometry. Primary OB cultures were positive for p75 at 70% (A), whereas, OB purified cultures were positive for p75 at 97% (B). At the same time, After 5 and 14 days in vitro cell surface expression of p75, respectively, in primary OM and purified OM cultures was determined by flow cytometry. Primary OM cultures were positive for p75 at 15% (C), whereas, OM purified cultures were positive for p75 at 98% (D). Numbers indicate the total population and the percentage of cells present in the indicated gated region.

Figure 3. Analysis of electrophysiological activities.

Measurement of cord dorsum potentials (A–C) shows that transplantation of OECs leads to increase potential amplitude from all OECs treated groups at all-time points studied (A–C). Md: Medium, OB: Olfactory Bulb, OM: Olfactory Mucosa. Statistical comparisons were performed using a nonparametric test (Kruskal-Wallis). Mean±SEM represents data from 10 rats per group. *: p value<0.05.

Figure 4. Analysis of locomotor activities.

A–C: horizontal activity D–F: vertical activity and G–I: vertical time. Measurement of locomotor activity (A–C) reveals that there is no significant difference between the groups for horizontal activity at all-time points. This analyses shows that transplantation of Primary OB and Purified OM improve vertical activity (F) and vertical time (I) at 60 days. Md: Medium, OB: Olfactory Bulb, OM: Olfactory Mucosa. Statistical comparisons were performed using a nonparametric test (Kruskal-Wallis). Mean±SEM represents data from 10 rats per group. *: p value<0.05.

Figure 5. Histological analyses.

Histological analyses on longitudinal sections were performed for neurofilament (A), GFAP (G), Neurocan (M) and ED1 (T). These analyses reveal that Primary OB, Purified OB and Purified OM OECs increase axonal regrowth (neurofilament) (A), reduce astrocyte reactivity (GFAP) (G) and glial scar formation (Neurocan) (M). At the same time this analysis reveals that OECs transplantations had no influence on monocytes/microglia infiltration (ED1) (T). Typical illustrations, are shown for each group for neurofilament (B–F), GFAP (H–L), neurocan (O–L) and ED1 (U–Y). Md: Medium, OB: Olfactory Bulb, OM: Olfactory Mucosa. Statistical comparisons were performed using a nonparametric test (Kruskal-Wallis). Mean±SEM represents data from 10 rats per group. *: p value<0.05; **: p value<0.01 and ***: p value<0.001.

Figure 6. MRI analyses.

MRI analyses revealed that Primary OB (C) and Purified OM OECs (D) decreased inflammatory infiltrate and edema 7 days after transplantation. Boxes represent areas of injury. Typical illustrations are shown for control (A), Md (B), Primary OB (C) and Purified OM OECs (D). Ctrl: Control, Md: Medium, OB: Olfactory Bulb, OM: Olfactory Mucosa. Illustrations are representative of four different animals for each group.

Figure 7. Tracking of GFP+ OM-OECs.

Tracking of GFP+ OM-OECs performed on longitudinal sections revealed that 30 (A–C) and 60 (D–F) days after transplantation, OECs could be found at the injury site (B and E) and both proximally (A and D) and distally (C and F) to the injection site. White arrows represent the lesion site. GFP/neurofilament co-staining shows that OECs are closely associated to neurofilament+ fibers (G–I2), 30 and 60 days after surgery. Scale bar = 100 µm. WH = White Matter and GH = Grey matter. Illustrations are representative of three different animals for each time point.