Successful Nerve Allografts Using Green Tea Polyphenol Solution and a Subtherapaeutic Dose of Fk506

INTRODUCTION Nerve allografts with immunosuppressants may be an alternative to conventional nerve autografts for the treatment of multiple nerve injuries with a long interstump gap. General use of immunosuppressants is associated with increased risks of infection, generation of neoplasm, and other toxic complications, some of which are lethal. It is therefore controversial to use peripheral nerve allografts with immunosuppressants when repairing peripheral nerve injuries. Green tea polyphenol protects tissues from ischemia, has antineoplasmic and anti-inflammatory effects, and suppresses immune responses. We have previously demonstrated successful nerve regeneration within peripheral nerve allografts that were immersed in a green tea polyphenol solution for one month. These results suggested that green tea polyphenol can protect nerve tissue from ischemia and suppress donor–host immune reactions during allotransplantation. However, a genomic study has shown at three months that only 14% of the cells originating from the donor nerves survived within transplanted nerve allografts that had been stored in the polyphenol solution. We hypothesized that more donor cells would survive within nerve allografts pretreated with polyphenol with the administration of a low dose of FK506, which is suitable for avoiding complications of the immunosuppressant. In this study, we have evaluated the immunosuppressive effect of the polyphenol extract combined with a subtherapeutic dose of FK506 in peripheral nerve allotransplantation with respect to allograft survival and nerve regeneration. METHODS A polyphenol mix extracted from green tea was purchased from PFI Inc. (Kyoto, Japan). It contained (–)-epigallo-catechin-3-O-gallate (28%), (–)-gallocatechin-3-O-gallate (11.6%), (–)-epicatechin-3-O-gallate (4.6%), (–)-epigallocatechin (15.0%), (+)-gallocatechin (14.8%), (–)epicatechin (7.0%) and (+)-catechin (9.5%), at a purity exceeding 90%. Twenty-millimeter sciatic nerve segments were excised from male Dark Agouti (DA) rats, stored in the polyphenol solution (1 mg/ml) for one month, and transplanted into recipient male Lewis rats to bridge 15 mm-long sciatic nerve gaps. The recipient rats were selected for major histocompatibility and complex mismatching. Animals were divided randomly into two groups. FK506 was injected subcutaneously at a rate of 0.2 mg/kg per day for the first group and at 2 mg/kg per day for the second group. The isograft control group had nerve segments harvested from male Lewis rats and immediately transplanted into other male Lewis rats. One week following transplantation, whole-blood samples were obtained from 10 rats in each of the three recipient groups, and the analysis for the T-cell CD4:CD8 ratio subsets was performed. Twelve weeks after surgery, electrophysiological and morphological studies were performed to assess the level of nerve regeneration within the transplanted nerve segments. A semiquantitative polymerase chain reaction (PCR) study specific for the sex-determining region of Ychromosome (Sry) was also performed to evaluate the amount of donor tissue in recipient rats. RESULTS The CD4:CD8 ratios in the polyphenol-0.2 allograft, polyphenol-2 allograft, and isograft groups were 2.79 ± 0.19, 2.75 ± 0.22 and 2.70 ± 0.14, respectively. There were no significant differences between the groups (P = 0.5978, 0.7927, 0.4311, respectively). Electrophysiological studies were performed at 12 weeks. The amplitudes of action potentials evoked in the pedal adductor muscle and the motor nerve conduction velocity (MNCV) in the operated limb were expressed as percentages of measurements taken from the contralateral nonoperated limb in each rat. The mean pedal adductor muscle amplitude of the polyphenol-0.2 allograft group (39.4 ± 6.4%) was similar to that of the polyphenol-2 allograft group (42.1 ± 11.1%, P = 0.5403) and that of the isograft group (37.1 ± 10.3%, P = 0.5841). The mean pedal adductor muscle amplitude of the polyphenol-2 allograft group was not significantly greater than that of the isograft group (P = 0.2506). The mean MNCVs (%) of the polyphenol-0.2 allograft group, the polyphenol-2 allograft group and the isograft group were 67.5 ± 9.2%, 67.9 ± 7.7% and 67.5 ± 12.3%, respectively (P = 0.9381, 0.9978, 0.9360, respectively). Morphological studies were subsequently performed at 12 weeks. There were significantly more myelinated axons in the polyphenol-0.2 allograft group (9,096 ± 1,583) and the polyphenol-2 allograft group (9,549 ± 1,147) than in the isograft group (6,411 ± 1,631, P = 0.0004, < 0.0001, respectively). There was no significant difference between the polyphenol-0.2 allograft group and the polyphenol-2 allograft group (P = 0.4964). There were no significant differences between the axon diameters in either the polyphenol-0.2 allograft group (1.57 ± 0.09), the polyphenol-2 allograft group (1.57 ± 0.11) or the isograft group (1.58 ± 0.09, P = 0.9456, 0.9095, 0.8557, respectively). The mean ratios of Sry to β-actin in the polyphenol-0.2 allograft, polyphenol-2 allograft, and isograft groups were 0.317 ± 0.075, 0.331 ± 0.09, and 0.622 ± 0.074, respectively, with no significant difference between the polyphenol-0.2 allograft group and in the polyphenol-2 allograft group (P = 0.6955). DISCUSSION There were no significant decreases in either the numbers of the CD4:CD8 ratio in the polyphenol-0.2 allograft group or the polyphenol2 allograft group in this study. Davenport et al. reported a significant decrease in the CD4:CD8 ratio of T-cell subsets in peripheral blood from days 2–3 to day 7 following transplantation in recipients with rejection episodes. Here we confirm, from the assessments of posttransplant blood lymphocyte phenotype subsets, that polyphenols combined with very low doses of FK506 reduce the donor–host immune reaction following nerve allotransplantation. The PCR studies showed that about 30% of the nerve cells in the graft were derived from polyphenol-treated nerve allografts plus 0.2 mg/kg per day FK506. Our previous study showed that 12 weeks following transplantation, 29% of the nerve cells in isografts and 14% of the cells in allografts originated from donor nerve segments treated by polyphenol without immunosuppressant. The current study has demonstrated successful peripheral nerve allograft and preservation using a combination of a subclinical dose of FK506 and green tea polyphenol pretreatment. Although our electrophysiological and morphohistological analyses found no significant differences in nerve regeneration between polyphenol-treated nerve allografts plus 0.2 mg/kg per day FK506 and those given 2 mg/kg per day FK506, nerve regeneration in the nerve segments treated with the FK506 administration was better than in nerve isografts without FK506. We believe that these results demonstrate a neurogenerative property for FK506. Our current study indicates a potential improvement in peripheral nerve allotransplantation in patients with peripheral nerve injuries with the use of low-dose immunosuppressants combined with polyphenol storage of the transplant segment. Moreover, our method simply immerses the peripheral nerve segments in a 1 mg/ml polyphenol solution at 4 °C and does not require any specialized equipment. However, further studies are necessary to investigate further andimprove the survival of donor neural cells. REFERENCES1. Ikeguchi R, Kakinoki R, Matsumoto T, et al. Successful storage ofperipheral nerve prior to transplantation using green tea polyphenol: anexperimental study in rats. Exp Neurol 2003; 184: 688.2. Ikeguchi R, Kakinoki R, Matsumoto T, et al. Peripheral nerveallograft stored in green tea polyphenol solution. Transplantation 2005;79: 688.3. Davenport M, Peakman M, Dunne JB, et al. Peripheral blood andintrahepatic subsets of T lymphocyte activation and function in liverallograft rejection and drug-induced tolerance in rats. Transpl Immunol