Administration of low-dose FK 506 accelerates histomorphometric regeneration and functional outcomes after allograft nerve repair in a rat model

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Summary

A substantial loss of peripheral nerves requires grafts for repair. In animal experiments, the use of allografts is successful only when rejection of the transplant is prevented and nerve regeneration is improved by the administration of the immunosuppressant FK 506 used in high doses. In this study, we examined the functional and morphometric outcome after allograft transplantation of the sciatic nerve in rats at low doses of FK 506. Functional recovery and quantitative assessment of myelination were investigated in un-operated controls, in rats receiving isograft transplants without FK 506 treatment and in rats receiving allograft transplants with FK 506 treatment (0.1 mg/kg and 0.2 mg/kg per day). Walking-track analysis at 4, 8, 12 and 16 weeks post-operation revealed significant functional recovery in allograft with FK 506 (0.1 mg/kg) compared with other groups, although levels of the un-operated controls were not reached. At 16 weeks, myelination of nerve sections from FK 506 (0.1 mg/kg)-treated and un-operated animals did not differ significantly. There was significantly less effect of the 0.2 mg/kg dose than of the 0.1 mg/kg dose, both in the histomorphological outcome and in the functional outcome. These findings indicate that higher doses of FK 506 are not necessary for nerve regeneration, and low-dose administration could be acceptable for clinical settings in future.

Introduction

Severe nerve lesions with loss of neural tissue require surgical repair to enable nerve regeneration. The use of grafts of autologous origin should achieve optimal results but has several important disadvantages such as the sacrifice of a healthy nerve from the patient, limited supply of donor nerves and mismatch in size between nerve and grafts (Oritguela et al., 1987, Rappaport et al., 1993). Because the supply of autografts is limited, the use of allografts is a promising alternative. However, the antigenicity of the graft tissue requires almost immunosuppressive therapy to avoid rejection and to enable regeneration. In this context, the use of immunosuppressive therapy is questionable because of the secondary risks and toxic effects of long-term and high-dosage immunosuppressant drugs (Neuhaus et al., 1994, Rifai et al., 2006).

Significant improvement in nerve regeneration rates could minimize denervation changes and improve long-term functional recovery from nerve injuries: the immunosuppressive drug FK 506 has been shown to have neuroprotective and neurotrophic actions in experimental models, increasing neurite elongation and accelerating the rate of nerve regeneration in vitro and in vivo (Lassner et al., 1989, Gold et al., 1995, Wang et al., 1997, Katsube et al., 1998, Doolabh and Mackinnon, 1999, Jost et al., 2000, Lee et al., 2000, Wang et al., 2002, Udina et al., 2002, Hontanilla et al., 2006). Thus, FK 506 could be useful, even clinically, for enhancing regeneration after surgical repair by improving the rate of axonal growth with allografts. In fact, there are reports of positive results regarding nerve regeneration in animals and humans immunosuppressed by FK 506 (Gold et al., 1994, Berger and Lassner, 1994, Mackinnon et al., 2001, Yang et al., 2003, Martin et al., 2005, Song et al., 2005, Snyder et al., 2006). Although previous studies found that FK 506 is maximally effective when administered in high doses (5–10 mg/kg/day) during the entire regeneration period in rat sciatic nerve models (Wang et al., 1997, Udina et al., 2004), prolonged systemic immunosuppression might not be justified for ensuring the success of nerve regeneration. It is, therefore, important to determine the optimal treatment dosage for FK 506 after allograft transplantation. In addition to supporting previous findings, this study examines the effect of low-dose FK 506 on nerve regeneration in a model more applicable to the severe peripheral-nerve injuries seen in clinical settings and more feasible to reduce the high doses of FK 506 accompanied with high rates of side-effects for a non-vital indication (Wang et al., 1997, Udina et al., 2004). Therefore, in this study choices of rat strains were made based on their nearly identical rat transplantion complexes (RTC), except for a variability in one of four haplotypes of the RT1 complex (RT1.C) (Günther and Walter, 2001). This is comparable to the necessary match of human leukocyte antigen (HLA) complexes before performing transplantations in humans. After the transplantation of kidneys between these strains, the survival time of the organs was >100 days without immunosuppression, whereas skin grafts showed massive rejection during this time (Stark et al., 1979).

Therefore, the findings presented in this study have the potential to affect significantly the clinical management of severe peripheral-nerve injuries.

Section snippets

Material and methods

All experiments were carried out in accordance with the National Institutes of Health (NIH) regulations for animal care and an animal use protocol was approved by the National Animal Care and Use Committee. Every effort was made to minimize the number of animals and their suffering.

Results

There were no animal drop outs during the examination period. All animals were healthy and showed no signs of automutilation or other signs of discomfort by the end of the study. Group I animals showed a median density of MBP per section field of 17.33 ± 3.80%, without significant differences among proximal, median or distal sections, an ASA of 45.02 ± 2.23° and a SFI of −3.22 ± 9.95%. All functional and morphometric data of the experimental groups II–V were set in relation to data of group I. There

Discussion

To describe the relationship between histomorphological and functional outcomes in this study, we applied an immunohistochemical method to mark myelinated nerve fibres. It is the regeneration and firing of myelinated nerve fibres which innervate the muscle (McNulty and Macefield, 2005, Miyasaka et al., 2007, Koob et al., 2007) that determine the walking tracks (ASA and SFI), not the total count of axons stained with different methods (Lyons et al., 1994, Archibald et al., 1995, Steuer et al.,

Acknowledgments

We are grateful to Peggy Radant, Angelika Stammler and Barbara Klazura, Brain Research Institute, University of Bremen, for technical assistance. This study was supported by grants from Astellas GmbH, Munich, Germany.

Conflict of interest statement: The authors have no financial or personal relationships with other people or organisations that could inappropriately influence their work.

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