In vivo studies

All in vivo studies were performed using adult rat model of sciatic nerve transection and repair (Kalbermatten et al., 2008). This is a widely used model for severe nerve transection studies and also used in several previous studies published from our laboratory (Timmer et al., 2003, Haastert et al., 2006a).

Although, this model also possess certain disadvantages e.g. rats start chewing the last three fingers of the operated side, denervated after sciatic nerve transection, a process called automutilation (autotomy). We also observed autotomy in some of the rats of our study in their left denervated paws.

A critical gap length is the minimum gap length which can prevent successful nerve regeneration after neuretmesis. 15 mm was reported as the critical gap length in the rat sciatic nerve transection model (Lundborg et al., 1982a, b). For our study with both ahSC and arSC, we selected a 10 mm gap length which was lesser than critical gap length and therefore was sufficient to obtain tissue cable regeneration for evaluation purposes over several time points ranging from 2-7

weeks. A 10 mm gap in the sciatic nerve was bridged using a silicone tube in the current study.

To prevent the rejection of the allograft (arSC in rat) or xenograft (ahSC in rat), immunosuppression was important. Cyclosporine and FK506 (Tacrolimus) are two drugs frequently used for immunosuppression studies in rat, but FK506 was reported to promote peripheral nerve regeneration in rats (Chen et al., 2008).

Therefore, cyclosporine was selected in a minimum required dose, which does not affect peripheral nerve regeneration in the dose of 10 mg / kg body weight / day without any associated side effects (Novartis guidelines). Subcutaneous or oral ways of administration were preferred using the drugs Sandimmun or Optoral (both cyclosporine), respectively. Both these drugs are known to exert the immunosuppressive effect by inactivating T and B-cells of the immune system (Novartis guidelines).

Optimal cell seeding in a nerve conduit is a core problem in tissue engineering.

An ideal nerve gap substitute would have to present an equally distributed number of cells that can activate the regrowing axons (Kalbermatten et al., 2008). It is therefore important to use optimal cell numbers in context of transplantation of adult human-derived primary cells (e.g. ahSC) because of three main reasons:

1. Limited availability.

2. Limited survival / proliferation capacities of these cells in vitro conditions.

3. Inappropriate and insufficient supplies of oxygen and other nutrients may lead to formation of toxic end products, which in turn inhibit peripheral nerve regeneration, in case of transplantation of excessive cell numbers in the lumen of the conduit.

In a previous study by Levi et al (1994), the number of ahSC used for transplantation in the sciatic nerve of rat was 16 x 10⁵ ahSC / animal, which was more than double the number we used in the study presented here (7 x 10⁵ ahSC

/ animal). Hence, total number of ahSC that are needed for a huge statistically relevant experiment would be usually very difficult to obtain from the limited supplies of human nerve biopsies. Moreover, in the light of results obtained in the current study, 7 x 10⁵ ahSC promoted peripheral nerve regeneration to the similar extent as reported in the mentioned study of Levi et al (1994). Therefore, 7 x 10⁵ ahSC were selected for all further in vivo transplantation experiments.

With reference to ahSC, two publications principally describe the identification and fate of these implanted ahSC after transplantation. Initial information about asymmetrical, but peripheral location of implanted LacZ-transduced ahSC (retroviral transduction) in the conduit was provided by Mosahebi et al (2001). In this report, transplanted SC nuclei were identified using LacZ staining.

Longitudinal tissue sections were used for quantifying axonal regeneration distance while transverse sections were primarily used for examining the association of transplanted SC and regenerating axons using LacZ-PanNF antibody co-staining, which demonstrated active axonal regeneration contributed by ahSC, 3 weeks post implantation.

Another report by Levi et al (1994) demonstrated by HNK-1 immunostaining (specific for human myelin) that implanted ahSC were capable of surviving, ensheathing and myelinating (to a very less extent) the rat axons when transplanted in a small gap of 8 mm in an immune deficient rat until 4 weeks post surgery. Later time points and longer gap lengths were not investigated in this study.

Detailed informations about the long time survival, myelination, distribution and in vivo behavior of implanted ahSC were needed over a longer time frame and across a larger gap length respectively, in order to get a clear picture of the cellular events occurring during axonal regeneration over different time points.

In this direction, the first step was to identify and locate the implanted ahSC.

Therefore, in the current study to track the implanted ahSC, they were labeled prior to implantation with PKH26-GL fluorescent membrane cell linker dye. With a

half life of more than 100 days, it has been reported to be effective in in vitro as well as in vivo cell tracking studies (Hugo et al., 1992).

In the current study, ahSC showed no altered survival after labeling in vitro when observed for 2 weeks post labeling. Accumulation of PKH26-GL not only in cell membrane but also in the cytoplasm of labeled cells is likely due to a previously described internalization of membrane areas and some dye leakage (Hugo et al., 1992). In the current study, we could also observe similar in vitro PKH26-GL dye leakage 2 weeks post labeling.

In contrast to Levi et al (1994), for transplanting ahSC / arSC in adult rats we focused on using a comparatively longer gap length of 10 mm over various time points (2-7 weeks). These experimental rats were immunosuppressed for the entire period of study (rather than being immunodeficient) for receiving allo and xenografts of arSC and ahSC, respectively. Further, nerve regeneration was analyzed with regard to immunohistochemistry (survival, myelination and in vivo behavior) and morphometry (tissue cable regeneration, axon number, diameter, cross sectional area, nerve fiber density, vascularization and ultrastructural demonstration).