Distribution pattern of PKH26-GL pre-labeled implanted ahSC

Fluorescent PKH26-GL positive cellular debris indicated dead pre-labeled cells in the lumen of the conduit that progressively got incorporated into the regenerated tissue.

An increase in density of GAP-43 positive regenerating axons suggested ongoing axonal regeneration.

Four weeks post surgery, PKH26-GL positive ahSC and debris appear to shift asymmetrically, towards one side of regenerated cable, which is likely due to force or push generated by the proliferating cells of host forming many Büngner bands in the lumen of nerve conduit.

It is already reported that Initial proliferation of perineurial cells and fibroblasts originating from host starts 3-4 days after injury and continues until some weeks depending upon the gap (Schröder et al., 1993).

Fluorescent debris appears to cause problem in the movement of the regenerating nerve fibers, 4 weeks post surgery. The surviving implanted ahSC, however, seem to support and promote the outgrowths from the mother axons originating from the proximal nerve stump (Jiang et al., 2007).

Until now there is no convincing literature to support the fate of cellular debris originating after transplantation of adult SC. Therefore, we can only speculate certain possibilities taking into account hints provided by some related reviews. It is likely that the release of growth factors from host and implanted SC together repel the debris to finally clear the way for the growing axons. There is another possibility that implanted ahSC together with host SC push the debris together to be digested by the secondary lysosomes so that this phagocytosis does not affect the surviving SC in the lumen of the conduit.

We found asymmetrical peripheral distribution of most living (PKH26-GL+ DAPI+) ahSC 6 weeks post surgery. Mosahebi et al (2001) also previously reported the peripheral distribution of nuclei of implanted ahSC which were transduced with LacZ. But, they investigated only 3 weeks after surgery. In this study, the rate of axonal regeneration into the PHB conduits graft was calculated from the proximal anastomosis and using immunostaining with PanNF. Additionally, the current study focused on details like survival and distribution of PKH26-GL pre-labeled implanted ahSC, number and type of nuclei present, arrangement and density of GAP-43+ fibers and fluorescent cellular debris present in the regenerated tissue cables after three different time points (2, 4 and 6 weeks post surgery).

There are several clinical reports in hematology regarding a possibility of immune incompatibility between xeno-transplanted and host cells in the graft (Holgersson, 2007, Ide et al., 2007). Therefore, we can also speculate that such an incompatibility also can exist between the transplanted ahSC and host arSC.

Six weeks after surgery enough host SC generated myelin could help in excluding surviving ahSC towards the peripheral areas. This possibility is confirmed from the observations made after transplantation of PKH26-GL labeled arSC in the same experimental setup. In this case, most of the surviving PKH26-GL positive arSC were found in the centre, unlike ahSC, which were found in most peripheral areas 6 weeks after implantation. This gives indication that in case of arSC, the host SC recognizes the implanted arSC as their `own´ (due to specific immune factors, MHC, expressed on their surface). There existed a possibility that they can also re-myelinate the axons. We could, however, not observe such remyelination as early as 6 weeks after surgery. Therefore, the probability of inter-species repulsion can not be excluded in context of the observations made in the current study.

After extensive microscopic observations, a close associationship was observed between surviving implanted ahSC and the regenerating nerve fibers in the centre most area of the regenerated tissue cable. However, only a few PKH26-GL positive ahSC displayed this associationship in the current study 6 weeks post surgery. Mosahebi et al (2001) also discussed such an associationship.

However, they could only demonstrate the associationship between the regenerating fibers and the transduced nuclei of transplanted ahSC, but not with the transplanted cell body.

This kind of associationship reflects several probable interactions between implanted ahSC and regenerating fibers, even when they did not display any remyelination capabilities. These might be:

1. Myelination may not be the principle function performed by implanted ahSC, but they might be involved in secretion of factors responsible for affecting growth of peripheral axons. Remyelination is rather performed mainly by the host arSC. The study by Levi et al. (1994) also supports this view and they demonstrated that ahSC isolated from peripheral nerves

are capable of myelination, but the myelin contribution of ahSC was much lesser in comparison of host arSC.

2. The close association between ahSC and regenerating nerve fibers can arise as a result from secretion of certain survival and growth factors from surviving ahSC, allowing the growing axons to successfully move towards the distal targets.

3. Cell-adhesion molecules expressed by the implanted ahSC (Dezawa and Nagano, 1996) could facilitate the growth, survival and target achievement of the regenerating fibers.

The latter two possibilities were evident also in the results of morphometrical analysis, where we found regenerated myelinated axons reaching the distal target in 50 % of the ahSC group animals, in comparison to acellular control group. Therefore, the associationship between regenerating nerve fibers and the surviving ahSC point towards a more supportive, nutritive and growth promoting function of the latter.

4.8 Correlation between morphometrical and histological data- potential