Cell Transplantation and Axonal Regeneration of Spinal Cord Axons

Although several studies suggest that transplantation of OECs into various spinal cord injury and demyelination

Figure 3 Remyelination after transplantation of hPAP OECs into the X-EB dorsal funiculus lesion.

(A) Low power plastic embedded section of the dorsal spinal cord 3 weeks after hPAP OEC transplantation.

(B) A higher power micrograph shows extensive myelinated profiles throughout the transplantation site. In adjacent frozen sections reacted for ALP, blue reaction product can be observed in the transplantation site (C). Higher power of this area shows numerous blue profiles characteristic of myelinated axons similar to that observed for SC transplantation in Fig. 2 (D). (E) Higher power images of plastic embedded sections shows myelinated axons many of which are associated with large cytoplasmic and nuclear surrounds characteristic of peripheral myelination. (F) These profiles are associated with hPAP reaction product (F). Scale bar in D; 200 |m in A, C; 25 |m in B, D; 7 |m in E, F. (Modified from Akiyama et al., 2004.)

Figure 3 Remyelination after transplantation of hPAP OECs into the X-EB dorsal funiculus lesion.

(A) Low power plastic embedded section of the dorsal spinal cord 3 weeks after hPAP OEC transplantation.

(B) A higher power micrograph shows extensive myelinated profiles throughout the transplantation site. In adjacent frozen sections reacted for ALP, blue reaction product can be observed in the transplantation site (C). Higher power of this area shows numerous blue profiles characteristic of myelinated axons similar to that observed for SC transplantation in Fig. 2 (D). (E) Higher power images of plastic embedded sections shows myelinated axons many of which are associated with large cytoplasmic and nuclear surrounds characteristic of peripheral myelination. (F) These profiles are associated with hPAP reaction product (F). Scale bar in D; 200 |m in A, C; 25 |m in B, D; 7 |m in E, F. (Modified from Akiyama et al., 2004.)

models can promote axonal regeneration, remyelination, and functional recovery (Franklin et al., 1996; Li et al., 1997, 1998; Ramon-Cueto et al., 1998, 2000; Imaizumi et al., 2000a, 2000b; Lu et al., 2002; Keyvan-Fouladi et al., 2003; Plant et al., 2003), Takami et al. (2002) reported that transplantation of SCs, but not OEC transplantation, results in improved hind-limb locomotor function in contusive spinal cord injury. Transplantation of OECs into spinal cord injury models mediates some degree of axonal regeneration and functional improvement even when transplantation is delayed (Lu et al., 2002; Keyvan-Fouladi et al., 2003). Myelinated axons spanning the lesion site have a characteristic peripheral pattern of myelination similar to that of SC myelination (Li et al., 1997, 1998; Imaizumi et al., 1998,

Figure 4 Conduction velocity measurements of demyelinated axons and after remyelination by Schwann cells or OECs. (A) Compound action potentials recorded from the dorsal columns (near midline and within 100 |m of the surface) with a glass microelectrode in vivo after stimulation of the dorsal column surface near the midline. Note an early and late negativity. When the surface of the spinal cord was washed with a 0 calcium and high (6 mM) magnesium Krebs' solution (2), the second negativity was eliminated indicating its synaptic nature. Thus the first negativity corresponds to conducting dorsal column axons. (B) Superimposed compound action potentials recorded at 1.0-mm increments longitudinally along the dorsal columns in normal (cont; 1), X-EB lesion (2), and 3 weeks after SC (3) and OEC (4) transplantation. (C) Histograms of conduction velocity (error bars indicate SEM) of dorsal column axons obtained from normal, 1, 3, and 6 weeks after EB injection without prior X-irradiation, after X-EB lesion induction, and 3 and 6 weeks after SC and OEC transplantation into the X-EB lesion. *P < 0.1, "P < 0.01, fP < 0.05, ffP < 0.05, and # not statistically significant. (Modified from Akiyama et al., 2004.)

2000a). Spinal cord injuries without OEC transplants can show limited SC-like myelination, presumably from invasion of the injury site from endogenous SCs (Brook et al., 1998; Imaizumi et al., 2000a, 2000b; Namiki et al., 2000; Takami et al., 2002) or possibly from precursor cells.

A unique feature associated with axonal regeneration after OEC transplantation is the occurrence of groups of axons within the transection lesion site with a peripheral pattern of myelination surrounded by a fibroblast-like cell that forms a "tunnel" around small clusters of myelinated axons (Li et al., 1997). An example of groupings of myeli-nated fibers surrounded by fibroblast-like cells is shown in Fig. 6. This section was taken from the center of the tran section zone indicating that the axons were regenerated. These tunnels have not been reported after SC transplantation into transection lesions (Imaizumi et al., 2000a, 2000b) or SC transplantation into demyelinating lesions (Lankford et al., 2002), suggesting that they are unique to OEC preparations. Li et al. (1998) referred to the surrounding fibroblast-like cells as "A" cells and the myelin-forming cells as "S" cells and suggest that both can be derived from the donor OECs. Although transplanted SCs can myelinate spinal cord axons (Blakemore and Crang, 1985; Baron-Van Evercooren et al., 1992; Honmou et al., 1996) and are associated with improved functional outcome (Takami et al., 2002), the "A" and "S" cell organization appears to be unique to OEC transplantation (Li et al., 1997, 1998; Imaizumi et al., 2000a, 2000b). However, Boyd et al. (2004) did not find LacZ-expressing "S" cells after transplantation of E18-derived OECs into a spinal cord compression injury model, but only LacZ-expressing fibroblast-like cells. They conclude that only the fibroblast-like cell ("A" cells) is derived from the OEC transplantation and that the "S" cells are exclusively derived from invading SCs.

We used an anti-GFP antibody in conjunction with immunoperoxidase staining on the electron microscopic level to identify the fate of donor GFP-expressing OECs transplanted in dorsal sectioned spinal cords of rat. We found evidence of GFP immunoreactivity on an ultrastruc-trual level in the cytoplasm of cells within the lesion site that were forming peripheral-like myelin (Sasaki et al., 2004). These data indicate that transplantation of adult OECs prepared at relatively high purity (>95% p75+) and not maintained in culture for extensive periods are able to form peripheral-like myelin around axons spanning a dorsal funiculus transection.

A difficulty in comparing results from OEC transplantation studies from various laboratories is that differences are present in the age of the animals used for cell harvesting, purification procedures, and lesion models into which the cells were transplanted. OECs used in our studies were prepared relatively acutely from the outer nerve layer of the adult olfactory bulb, an area rich in OECs in vivo (Devon and Doucette, 1992). The degree of cell purity (>95%) in our cell suspension as assessed using p75/S100 immuno-staining was about the same as in other studies where immunopanning techniques were used (Takami et al., 2002; Lakatos et al., 2003) or where OECs were prepared from embryonic tissue (Devon and Doucette, 1992). Mitotic inhibitors and stimulators of cell proliferation and differentiation were used in those studies. In our cell preparation method from adult tissue, we did not use mitotic inhibitors, nor did we stimulate proliferation and differentiation in vivo. Contamination by SCs, which are also p75/S100 positive, in our cultures would be problematic in the interpretation that adult OECs are able to form peripheral-like myelin. However, one would expect at best a very minor contamina

Figure 5 Transplantation of transgenic pig OECs into the demyelinated monkey spinal cord. (A) Low-power micrograph of lesion after cell transplantation in the dorsal funiculus (DF). Remyelination was observed within the white dashed lines and most of the dorsal funicular region outside of the dashed lines remained demyelina-tion. (B) The central core of the lesion was densely remyelinated. (C) The boxed area in (B), showing myelinated axon profiles exhibiting a peripheral pattern of remyelination. (D) The edge of the densely remyelinated zone a transition from demyelinated (left) to remyelinated axons can be seen. Scale bar, (a) = 1.25 mm, (c) = 50 |m, (b, d) = 125 |m. (Modified from Radtke et al., 2004.)

Figure 5 Transplantation of transgenic pig OECs into the demyelinated monkey spinal cord. (A) Low-power micrograph of lesion after cell transplantation in the dorsal funiculus (DF). Remyelination was observed within the white dashed lines and most of the dorsal funicular region outside of the dashed lines remained demyelina-tion. (B) The central core of the lesion was densely remyelinated. (C) The boxed area in (B), showing myelinated axon profiles exhibiting a peripheral pattern of remyelination. (D) The edge of the densely remyelinated zone a transition from demyelinated (left) to remyelinated axons can be seen. Scale bar, (a) = 1.25 mm, (c) = 50 |m, (b, d) = 125 |m. (Modified from Radtke et al., 2004.)

tion of SCs, possibly associated with blood vessel innervation (Takami et al., 2002). Such minor contamination could not account for the vast majority (>95%) of our cells displaying a p75/S100 phenotype. Yet, the issue of cell contamination with SCs or meningeal cells (Lakatos et al., 2003) is important and clearly should be addressed in future studies.

The mechanisms for the functional improvement observed after OEC transplantation into spinal cord injury models are not clear and have been suggested to include long-tract regeneration (Li et al., 1997; Ramon-Cueto et al., 2000), axonal sparing and neuroprotection (Plant et al., 2003), sprouting and plasticity associated with novel poly-synaptic pathways (Keyvan-Fouladi et al., 2003), recruitment of endogenous SCs (Takami et al., 2002; Boyd et al.,

2004), and remyelination (Franklin et al., 1996; Imaizumi et al., 2000a, 2000b). OECs secrete a number of trophic factors such as nerve growth factor, glial-derived neurotrophic factor, brain-derived neurotrophic factor, and ciliary neu-rotrophic factor that could contribute to these events (Woodhall et al., 2001). It is possible that more than one of these mechanisms is operative.

OECs have emerged as an important cell candidate for cell transplantation strategies to improve functional outcome in adult spinal cord injury. Clinical investigations in spinal cord injury using OEC engraftment are in progress (Senior, 2002; Huang et al., 2003). Although it is generally agreed that, under appropriate cell preparation and transplantation conditions, functional outcome can be enhanced by OEC transplantation, questions still remain with regard to the

Figure 6 Semithin plastic sections stained with methylene blue/Azure II through the transection site 5 weeks after transplantation of OECs. Note the clustering of myelinated axons surrounded by a cellular element. These surrounding cells are not observed after transplantation of SCs and appear to be unique to OEC transplantation. Scale bar = 8 |m.

Figure 6 Semithin plastic sections stained with methylene blue/Azure II through the transection site 5 weeks after transplantation of OECs. Note the clustering of myelinated axons surrounded by a cellular element. These surrounding cells are not observed after transplantation of SCs and appear to be unique to OEC transplantation. Scale bar = 8 |m.

in vivo fate of transplanted OECs. Determination of the relative contribution of cellular repair such as remyelination vs. trophic support for endogenous recruitment of cells, neuroprotection, and synaptic plasticity by OECs will be important for a comprehensive evaluation of the potential therapeutic efficacy of OECs as a cell therapy in spinal cord injury. As our understanding of potential trophic influences of OECs is expanded, this could suggest novel pharmacological approaches to the treatment of spinal cord injury.

Another aspect of spinal cord injury is the reduction in cyclic adenosine monophosphate (cAMP) in injured neurons (Pearse et al., 2004). Increased cAMP levels in growth cones has been shown to enable neurons to extent neurites across inhibitory substrates such as myelin (Cai et al., 2001). One study demonstrated the combined transplantation of SCs with inhibition of cAMP hydrolysis (Rolipram treatment) and db-cAMP treatment promoted axonal growth and functional recovery after spinal cord injury (Pearse et al., 2004). This study emphasizes that a combination of cell therapy and pharmacological approaches may maximize functional recovery in spinal cord injury.

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